Toll-like receptor 5 ligands and methods of use

ABSTRACT

The invention provides an immunomodulatory flagellin peptide having substantially the same amino acid sequence GALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQ (SEQ ID NO:44), or a modification thereof, and having toll like receptor 5 (TLR5) binding. The immunomodulatory flagellin peptide also can have substantially the same amino acid sequence TQFSGVKVLAQDNTLTIQVGANDGETIDIDLKQINS QTLGLDTL (SEQ ID NO:45); EGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVNG (SEQ ID NO:46) or MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANF TANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQS (SEQ ID NO:47), or a modification thereof. Also provided is an immunomodulatory flagellin peptide having substantially the same amino acid sequence LQKIDAALAQVDTLRSDLGAVQNRFNSAITNL (SEQ ID NO:48), or a modification thereof, and having toll like receptor 5 (TLR5) binding. The immunomodulatory flagellin peptide also can have substantially the same amino acid sequence TLRSDLGAVQNRFNSAITNLGNTVNNLSS (SEQ ID NO:49) or EQAAKTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLSS (SEQ ID NO:50), or a modification thereof. Further provided is an immunomodulatory flagellin peptide having substantially the same amino acid sequence GALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAE ITQ (SEQ ID NO:44) and substantially the same amino acid sequence LQKIDAALAQVDTLRSDLGAVQNRFNSAITNL (SEQ ID NO:48), or a modification thereof, and having toll like receptor 5 (TLR5) binding. Methods of using immunomodulatory flagellin peptides additionally are provided.

This application is a continuation-in-part of 10/125,692, filed Apr. 17,2002, and is based on, and claims the benefit of, U.S. ProvisionalApplication No. 60/285,477, filed Apr. 20, 2001, both of which areincorporated herein by reference.

This invention was made with government support under grant numbers5R37AI025032 and 5R01AI032972, awarded by the National Institutes ofHealth. The United States Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death in the United States,accounting for one in every four deaths. This year, it is expected thatover 1500 Americans will die of cancer each day and that a million newcases of cancer will be diagnosed. The most common treatments for cancerare surgery, radiation and chemotherapy. According to the AmericanCancer Society, immunotherapy can be considered as the “fourth modality”in the treatment of cancer. Immunotherapy is treatment that stimulatesone's own immune system to fight cancer.

Cancer is a group of diseases characterized by uncontrolled growth ofabnormal cells of the body. All types of cancer involve the malfunctionof genes that control cell growth and division. Some of these genesbecome incorrectly regulated, resulting in over- or under-production ofa particular protein, while others become mutated, resulting in unusualor abnormal proteins that alter normal cellular functions. Theseabnormal proteins, referred to as “tumor cell antigens,” should berecognized and destroyed by an individual's immune system as “foreign”antigens.

However, the immune system of a cancer patient may ignore these tumorantigens and be unresponsive to the growing tumor. Using immunotherapyapproaches, such as cancer vaccines and immune system modulators, anindividual's immune system can be induced to mount a potent immuneresponse against tumor cell antigens, resulting in elimination of cancercells. A cancer vaccine can contain a tumor cell antigen that stimulatesthe immune system to recognize and destroy cells which display thatantigen. Treating an individual with such a cancer vaccine can result ina humoral response, which involves producing antibodies that recognizeand target tumor cells for destruction and a cellular response, whichinvolves producing cytotoxic T cells that recognize and destroy tumorcells directly, or both responses. It can be desirable to obtain both ahumoral and cellular immunity response during immunotherapy because botharms of immune response have been positively correlated with beneficialclinical responses. To help stimulate either or both humoral andcellular immune responses, a cancer vaccine can be combined with anadjuvant, which is a substance that stimulates a general immuneresponse.

The potency of cancer vaccines is greatly enhanced by the use ofadjuvants. The selection of an adjuvant for use with a particularvaccine can have a beneficial effect on the clinical outcome ofvaccination. Some vaccines are ineffective in the absence of anadjuvant. Effectiveness of a vaccine may be particularly troublesomewhen the vaccine is produced from self antigens such as those requiredfor cancer vaccines or other non-infectious disease vaccines. In view ofthe beneficial effects of adjuvants in vaccine formulations, it issurprising that only one type of adjuvant, aluminum-salt basedadjuvants, are currently in wide use in United States-licensed vaccines.

Thus, there exists a need for more and improved immunological adjuvants.The present invention satisfies this need and provides relatedadvantages as well.

SUMMARY OF THE INVENTION

The invention provides an immunomodulatory flagellin peptide havingsubstantially the same amino acid sequenceGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQ (SEQ ID NO:44), or amodification thereof, and having toll like receptor 5 (TLR5) binding.The immunomodulatory flagellin peptide also can have substantially thesame amino acid sequence TQFSGVKVLAQDNTLTIQVGANDGETIDIDLKQINS QTLGLDTL(SEQ ID NO:45); EGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVNG (SEQID NO:46) or MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQS (SEQ ID NO:47), or amodification thereof. Also provided is an immunomodulatory flagellinpeptide having substantially the same amino acid sequenceLQKIDAALAQVDTLRSDLGAVQNRFNSAITNL (SEQ ID NO:48), or a modificationthereof, and having toll like receptor 5 (TLR5) binding. Theimmunomodulatory flagellin peptide also can have substantially the sameamino acid sequence TLRSDLGAVQNRFNSAITNLGNTVNNLSS (SEQ ID NO:49) orEQAAKTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLSS (SEQ ID NO:50), or amodification thereof. Further provided is an immunomodulatory flagellinpeptide having substantially the same amino acid sequenceGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAE ITQ (SEQ ID NO:44) andsubstantially the same amino acid sequenceLQKIDAALAQVDTLRSDLGAVQNRFNSAITNL (SEQ ID NO:48), or a modificationthereof, and having toll-like receptor 5 (TLR5) binding. Methods ofusing immunomodulatory flagellin peptides additionally are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows NF-kB activation and TNFa production in cells expressingCD4-TLR4 or CD4-TLR5.

FIG. 2 shows selective induction of TLR5-stimulated activation of NF-kBby P. aeruginosa and L. monocytogenes cultures compared to LPS andlipopeptide.

FIG. 3 shows the purification of a TRL5-stimulating activity from L.monocytogenes culture supernatant.

FIG. 4 shows the identification by mass spectrometry of flagellin as aTLR5-stimulating activity.

FIG. 5 shows that flagellin expression in bacteria reconstitutesTLR5-stimulating activity.

FIG. 6 shows systemic induction of IL-6 in wild type mice treated withpurified flagellin.

FIG. 7 shows a comparison of flagellin amino acid sequences from 22species of bacteria and a consensus sequence of amino acid residuesconserved across species.

FIG. 8 shows that the D1 domain N- and C-terminal abrogates TLR5recognition.

FIG. 9 shows that TLR5 recognizes discrete site in the D1 domain.

FIG. 10 shows that flagellin alanine point mutations reduce TLR5recognition.

FIG. 11 shows that TLR5 recognizes monomeric rather than filamentousflagellin.

FIG. 12 shows that biotinylated flagellin specifically associates withTLR5.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to flagellin derived peptides that exhibitimmunomodulatory activity and to methods of inducing an immune responsethrough activation of toll-like receptor 5 (TLR5). The identification ofactive flagellin peptides and their corresponding receptor, TLR5,expands the available treatment methods for inducing an immune response.Moreover, the identification of active flagellin peptides and theircognate receptor allows the identification of immunomodulatorycompounds.

In one embodiment, the invention is directed to immunomodulatoryflagellin peptides that bind to TLR5 and induce a TLR5-mediatedactivity. The peptides can be used, for example, to effectivelystimulate an immune response or ameliorate a pathological condition byadministration of immunomodulatory flagellin peptides and combinationsof such peptides with antigens and other immunomodulatory molecules.Full length flagellin polypeptides are also used in the methods of theinvention to stimulate an immune response. An advantage of theimmunomodulatory flagellin peptides of the invention is that theyprovide the specificity of flagellin together with the availability ofrapid and efficient methods for recombinant and chemical synthesis ofpeptides. The immunomodulatory flagellin peptides of the invention cantherefore be combined with numerous well known modes of administrationfor the treatment of a wide variety of pathological conditions.

In another embodiment, the invention provides a method of inducing animmune response in an individual by administering a vaccine containingan immunomodulatory flagellin peptide of the invention and an antigen.An immunomodulatory flagellin peptide of the invention functions tostimulate an innate immune response. The innate immune response involvesthe production of immunomodulatory molecules that beneficially promotethe adaptive immune response. The adaptive immune response includes bothhumoral and cell-mediated immune responses to antigen. Thus, a flagellinpeptide functions to boost either or both humoral and cell-mediatedimmune responses against the antigen. A boost in an immune responsecauses a general increase in immune system activity that can result inthe destruction of foreign or pathologically aberrant cells thatotherwise could have escaped the immune response.

As used herein, the term “flagellin” is intended to mean a flagellinpolypeptide contained in a variety of Gram-positive or Gram-negativebacterial species. The nucleotide and amino acid sequences of flagellinfrom 22 bacterial species are depicted in FIG. 7. The nucleotidesequences encoding the listed flagellin polypeptides are publicallyavailable in the NCBI Genbank database. The flagellin sequences fromthese and other species are intended to be encompassed by the termflagellin as used herein. Therefore, the sequence differences betweenspecies is included within the meaning of the term.

As used herein, the term “peptide” is intended to mean two or more aminoacids covalently bonded together. The term “flagellin peptide” isintended to mean a peptide or fragment encoded by a portion of thenucleotide sequence or having a portion of the amino acid sequence whichexhibits substantially the same sequence identity to the flagellinsequences as described above and identified in FIG. 7 and binds totoll-like receptor 5 (TLR5). For example, a flagellin peptide amino acidsequence is about 65% or greater in sequence identity to a portion ofthe S. Typhimurium1 sequence, GAVQNRFNSAIT, identified as SEQ ID NO:2,encoded by the nucleic acid sequence identified as SEQ ID NO:1.Therefore, flagellin peptides having amino acid substitutions that donot substantially alter TLR5 binding are included within the definitionof a flagellin peptide. For example, flagellin peptides which containone or more alanine substitutions and have substantially the same TLR5binding activity as the flagellin peptide identified as SEQ ID NO:2 areincluded within the definition of a flagellin peptide. Exemplaryflagellin peptides containing alanine substitutions and havingsubstantially the same TLR5 binding activity as the flagellin peptideidentified as SEQ ID NO:2 include, for example, GAVANRFNSAIT (SEQ IDNO:3) and GAVQNAFNSAIT (SEQ ID NO:4). Flagellin peptides consisting ofgreater than twelve amino acids and having TLR5 binding activity cansimilarly contain amino acid substitutions, so long as such substitutedpeptides retain substantially the same TLR5 binding activity. Examplesof such flagellin peptides containing substitutions of various aminoacid residues with alanine include ADTRDLGAVQNRFNSAIT (SEQ ID NO:37),VDARDLGAVQNRFNSAIT (SEQ ID NO:38) and VDTADLGAVQNRFNSAIT (SEQ ID NO:39).A flagellin peptide of the invention does not include a full lengthflagellin polypeptide. A flagellin peptide is intended to includemolecules which contain, in whole or in part, non-amide linkages betweenamino acids, amino acid analogs and mimetics. Similarly, a flagellinpeptide also includes cyclic peptides and other conformationallyconstrained structures. A flagellin peptide of the invention includespolypeptides having several hundred or more amino acid residues and cancontain a heterologous amino acid sequence.

The term flagellin peptide specifically excludes fragments of flagellindescribed in Newton et al. Science, 244: 70-72 (1989); Kuwajima, G., J.Bacteriol. 170: 3305-3309 (1988); McSorley et al., J. Immunol. 164:986-993(2000); and Samatey et al. J. Struct. Biol. 132: 106-111 (2000).

As used herein, term “immunomodulatory flagellin peptide,” is intendedto mean a peptide or fragment having a portion of the amino acidsequence which exhibits substantially the same sequence identity to theflagellin sequences as described above and shown in FIG. 7 and binds totoll-like receptor 5 (TLR5). For example, an immunomodulatory flagellinpeptide amino acid sequence is about 65% or greater in sequence identityto a portion of the S. Typhimurium1 sequence, GAVQNRFNSAIT, identifiedas SEQ ID NO:2, encoded by the nucleic acid sequence identified as SEQID NO:1. Therefore, immunomodulatory flagellin peptides having aminoacid substitutions that do not substantially alter TLR5 binding areincluded within the definition of an immunomodulatory flagellin peptide.For example, immunomodulatory flagellin peptides which contain one ormore alanine substitutions and have substantially the same TLR5 bindingactivity as the flagellin peptide identified as SEQ ID NO:2 are includedwithin the definition of a flagellin peptide. Exemplary immunomodulatoryflagellin peptides containing alanine substitutions and havingsubstantially the same TLR5 binding activity as the flagellin peptideidentified as SEQ ID NO:2 include, for example, GAVANRFNSAIT (SEQ IDNO:3) and GAVQNAFNSAIT (SEQ ID NO:4). Immunomodulatory flagellinpeptides consisting of greater than twelve amino acids and having TLR5binding activity can similarly contain amino acid substitutions, so longas such substituted peptides retain substantially the same TLR5 bindingactivity. Examples of such immunomodulatory flagellin peptidescontaining substitutions of various amino acid residues with alanineinclude ADTRDLGAVQNRFNSAIT (SEQ ID NO:37), VDARDLGAVQNRFNSAIT (SEQ IDNO:38) and VDTADLGAVQNRFNSAIT (SEQ ID NO:39). An immunomodulatoryflagellin peptide of the invention does not include a full lengthflagellin polypeptide. An immunomodulatory flagellin peptide is intendedto include molecules which contain, in whole or in part, non-amidelinkages between amino acids, amino acid analogs and mimetics.Similarly, an immunomodulatory flagellin peptide also includes cyclicpeptides and other conformationally constrained structures. Animmunomodulatory flagellin peptide of the invention includespolypeptides having several hundred or more amino acid residues and cancontain a heterologous amino acid sequence.

An immunomodulatory flagellin peptide, polypeptide or modificationthereof, of the invention binds to toll-like receptor 5 (TLR5) andinduces a TLR5-mediated response. It is understood that minormodifications can be made without destroying the TLR5 binding activity,TLR5-mediated response stimulating activity or immune responsemodulating activity of an flagellin peptide or polypeptide and that onlya portion of the primary structure may be required in order to effectactivity. Such modifications are included within the meaning of theterms flagellin polypeptide and flagellin peptide so long as TLR5binding activity, TLR5 response stimulating or immune responsestimulating activities are retained. Further, various molecules can beattached to flagellin polypeptides and peptides, including for example,other polypeptides, carbohydrates, nucleic acids or lipids. Suchmodifications are included within the definition of the term.

Minor modifications of flagellin polypeptide and peptides having atleast about the same TLR5 binding activity, TLR5 response stimulating orimmune response stimulating activity as the referenced polypeptide orpeptide include, for example, conservative substitutions of naturallyoccurring amino acids and as well as structural alterations whichincorporate non-naturally occurring amino acids, amino acid analogs andfunctional mimetics. For example, a Lysine (Lys) is considered to be aconservative substitution for the amino acid Arg. Similarly, a flagellinpeptide containing mimetic structures having similar charge and spacialarrangements as reference amino acid residues would be considered amodification of the reference polypeptide or peptide so long as thepeptide mimetic exhibits at least about the same activity as thereference peptide.

As used herein, the term “amino acid” is intended to mean both naturallyoccurring and non-naturally occurring amino acids as well as amino acidanalogs and mimetics. Naturally occurring amino acids include the 20(L)-amino acids utilized during protein biosynthesis as well as otherssuch as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine,homocysteine, citrulline and ornithine, for example. Non-naturallyoccurring amino acids include, for example, (D)-amino acids, norleucine,norvaline, p-fluorophenylalanine, ethionine and the like. Amino acidanalogs include modified forms of naturally and non-naturally occurringamino acids. Such modifications can include, for example, substitutionor replacement of chemical groups and moieties on the amino acid or byderivitization of the amino acid. Amino acid mimetics include, forexample, organic structures which exhibit functionally similarproperties such as charge and charge spacing characteristic of thereference amino acid. For example, an organic structure which mimicsArginine (Arg or R) would have a positive charge moiety located insimilar molecular space and having the same degree of mobility as thee-amino group of the side chain of the naturally occurring Arg aminoacid. Mimetics also include constrained structures so as to maintainoptimal spacing and charge interactions of the amino acid or of theamino acid functional groups. Those skilled in the art know or candetermine what structures constitute functionally equivalent amino acidanalogs and amino acid mimetics.

Specific examples of amino acid analogs and mimetics can be founddescribed in, for example, Roberts and Vellaccio, The Peptides:Analysis, Synthesis, Biology, Eds. Gross and Meinhofer, Vol. 5, p. 341,Academic Press, Inc., New York, N.Y. (1983), the entire volume of whichis incorporated herein by reference. Other examples include peralkylatedamino acids, particularly permethylated amino acids. See, for example,Combinatorial Chemistry, Eds. Wilson and Czarnik, Ch. 11, p. 235, JohnWiley & Sons Inc., New York, N.Y. (1997), the entire book of which isincorporated herein by reference. Yet other examples include amino acidswhose amide portion (and, therefore, the amide backbone of the resultingpeptide) has been replaced, for example, by a sugar ring, steroid,benzodiazepine or carbo cycle. See, for instance, Burger's MedicinalChemistry and Drug Discovery, Ed. Manfred E. Wolff, Ch. 15, pp. 619-620,John Wiley & Sons Inc., New York, N.Y. (1995), the entire book of whichis incorporated herein by reference. Methods for synthesizing peptides,polypeptides, peptidomimetics and proteins are well known in the art(see, for example, U.S. Pat. No. 5,420,109; M. Bodanzsky, Principles ofPeptide Synthesis (1st ed. & 2d rev. ed.), Springer-Verlag, New York,N.Y. (1984 & 1993), see Chapter 7; Stewart and Young, Solid PhasePeptide Synthesis, (2d ed.), Pierce Chemical Co., Rockford, Ill. (1984),each of which is incorporated herein by reference).

As used herein, the term “immune response” is intended to mean to ameasurable or observable reaction to an antigen or immunomodulatorymolecule mediated by one or more cells of the immune system. An immuneresponse begins with an antigen or immunomodulatory molecule binding toan immune system cell and terminates with destruction of antigen andcells containing antigen or alteration in immune cell function. Areaction to an antigen or immunomodulatory molecule is mediated by manycell types, including a cell that initially binds to an antigen orimmunomodulatory molecule and cells that participate in mediating aninnate, humoral, cell-mediated immune response. An innate immuneresponse involves binding of pathogen-associated molecular patterns(PAMPs) to cell surface receptors, such as toll-like receptors.Activation of toll-like receptors in response to PAMPs leads to theproduction of immunomodulatory molecules, such as cytokines andco-stimulatory molecules, that induce an immune response. A humoralresponse involves interaction of B cells with antigen and B celldifferentiation into antibody-secreting cells. A cell-mediated responseinvolves various subpopulations of T cells that recognize antigenpresented on self-cells, including helper T cells that respond toantigen by producing cytokines and cytotoxic T cells that respond toantigen by developing into cytotoxic T lymphocytes, which mediatekilling of altered self-cells. The term immune response includesmeasurable or observable reactions produced by any cell type thatparticipates in the processes through which immune system cells areactivated and antigen containing cells are destroyed. Such measurablereactions include, for example, production of immunomodulatorymolecules, migration and proliferation.

An “immunomodulatory molecule” is a molecule that alters an immuneresponse. An immunomodulatory molecule can be, for example, a compound,such as an organic chemical; a polypeptide, such as an antibody orcytokine; a nucleic acid, such as a DNA or RNA molecule; or any othertype of molecule that alters an immune response. An immunomodulatorymolecule can alter an immune response by directly or indirectly alteringan activity of a cell that mediates an immune response. Animmunomodulatory molecule can act directly on an immune system cell, forexample, by binding to a cell surface receptor and stimulating orinhibiting proliferation, differentiation, or expression, secretion orreceptor binding of immune system regulatory molecules such asco-stimulatory receptors and ligands, cytokines, and chemokines.Examples of naturally occurring molecules that act directly on immunesystem cells to alter an immune response include PAMPs, cytokines,chemokines and growth factors. Other examples of molecules that actdirectly on immune system cells to alter an immune response includemolecules that alter receptor functions, such as antibodies toreceptors, soluble cytokine receptors, receptor agonists andantagonists, molecules that alter the production of immunomodulatorymolecules, such as inhibitors of converting enzymes and moleculesinvolved in the intracellular transport and secretion ofimmunomodulatory molecules.

An immunomodulatory molecule can indirectly alter the activity of aparticular immune system cell by altering the amount or activity of amolecule that regulates a cellular activity of the cell. For example, acytokine, chemokine, or growth factor produced by an immune system cell,such as a macrophage, can stimulate or inhibit various cellularactivities of B and T lymphocytes. Immune cell functions that can bestimulated or inhibited by an immunomodulatory molecule include, forexample, immune cell activation, co-activation, proliferation,production of cytokines, cellular interactions and migration. Animmunomodulatory molecule can therefore act on a variety of immune celltypes and can alter a variety of cellular functions. An immunomodulatoryflagellin peptide, polypeptide or modifications thereof used in themethods of the invention are examples of immunomodulatory moleculesuseful for inducing an immune response, for example, by binding to TLR5and inducing a TLR5-mediated increase in macrophage production of TNFa,IL-1 and IL-6. The flagellin polypeptides, peptides and modificationsthereof, are also useful for indirectly inducing an immune responsebecause immunomodulatory molecules produced by a TLR5-expressing cell inresponse to flagellin will alter the activities of immune system cellsthat respond to the particular immunomodulatory molecules produced.

An immunomodulatory molecule can mediate an immune response that isspecific for a target antigen or nonspecific. A specificimmunomodulatory molecule alters an immune response to a particulartarget antigen. Examples of specific immunomodulatory molecules includemonoclonal antibodies, including naked monoclonal antibodies, drug-,toxin- or radioactive compound-conjugated monoclonal antibodies, andADCC targeting molecules. Such immunomodulatory molecules stimulate animmune response by binding to antigens and targeting cells fordestruction. An immunomodulatory molecule can be used to suppress animmune response to an antigen. For example, a tolerogenizing moleculecan be used to suppress an immune response to a self-antigen.

Nonspecific immunomodulatory molecules stimulate or inhibit the immunesystem in a general manner through various mechanisms that can include,for example, stimulating or suppressing cellular activities of immunesystem cells. Nonspecific immunomodulatory molecules useful forstimulating an immune responses include, for example, agents thatstimulate immune cell proliferation, immune cell activation andproduction of cytokines and co-stimulatory molecules. Well knownimmunomodulatory molecules that stimulate an immune response are, forexample, interleukins, interferons, levamisole and keyhole limpethemocyanin. Nonspecific immunomodulatory molecules useful forsuppressing immune responses include, for example, agents that inhibitcytokines synthesis or processing, specific cytokine receptor blockingreagents such as soluble receptors and receptor antagonists, andcytokines that down-regulate or inhibit the production of otherimmunomodulatory molecules. Well known immunomodulatory molecules forsuppressing an immune response include, for example, cyclosporin,rapamycin, tacrolimus, azathioprine, cyclophosphamide and methotrexate.

Immunomodulatory molecules can be contained in a mixture of molecules,including a natural or man-made composition of molecules. Exemplarynatural compositions of immunomodulatory compounds include, for example,those contained in an organism such as Bacille Calmette-Guerin (BCM) orCorynbacterium parvum. Exemplary man-made compositions ofimmunomodulatory molecules include, for example, QS-21, DETOX andincomplete Freund's adjuvant.

As used herein, the term “adjuvant” when used in reference to a vaccine,is intended to mean a substance that acts generally to accelerate,prolong, or enhance the quality of specific immune responses to avaccine antigen. An adjuvant can advantageously reduce the number ofimmunizations or the amount of antigen required for protectiveimmunization.

As used herein, the term “antigen-specific immune response” is intendedto mean a reaction of one or more cells of the immune system to aparticular antigen that is not substantially cross-reactive with otherantigens.

As used herein, the term “antigen” is intended to mean a molecule whichinduces an immune response. An antigen can be a crude mixture ofmolecules, such as a cell, or one or more isolated molecules. Examplesof crude antigens include attenuated organisms, inactivated organisms,viral particles and tumor cells. Examples of isolated antigens include apolypeptide, lipoprotein, glycoprotein, lipid, anti-idiotype antibody,toxoid, polysaccharide, capsular polysaccharide and nucleic acid. Suchisolated antigens can be naturally occurring, recombinantly produced, orsynthesized. Exemplary naturally occurring antigens include purifiedmicrobial macromolecules. Exemplary recombinantly produced antigensinclude cloned microbial and tumor cell antigens. Exemplary synthesizedantigens include synthetic peptides and nucleic acids.

As used herein, the term “vaccine” is intended to mean a compound orformulation which, when administered to an individual, stimulates animmune response against an antigen. A vaccine is useful for preventingor ameliorating a pathological condition that will respond favorably toimmune response modulation. A vaccine can contain isolated or crudeantigen, and can contain one or more antigens. A vaccine can contain oneor more adjuvants.

As used herein, the term “immunogenic amount” is intended to mean anamount of an immunomodulatory flagellin polypeptide, peptide ormodifications thereof, or combinations thereof with one or moremolecules, such as an antigen or other immunomodulatory molecule,required to effect an immune response. The dosage of an immunomodulatoryflagellin polypeptide, peptide, or modifications thereof, independentlyor in combination with one or more molecules, will depend, for example,on the pathological condition to be treated, the weight and condition ofthe individual and previous or concurrent therapies. The appropriateamount considered to be an immunogenic dose for a particular applicationof the method can be determined by those skilled in the art, using theguidance provided herein. For example, the amount can be extrapolatedfrom in vitro or in vivo assays as described below. Those skilled in theart will understand that the condition of the patient needs to bemonitored through the course of therapy and that the amount of thecomposition that is administered can be adjusted according to patientresponse to therapy.

The term “pathologically aberrant cell” is intended to mean a cell thatis altered from a normal physiological or cellular state. Suchalteration can be due to changes in physiology or phenotype associatedwith a disease or abnormal condition of a mammalian cell or tissue.Pathologically aberrant cells include cells lacking normal control ofcellular functions, such as growth, differentiation, and apoptosis,resulting in altered gene and protein expression. Cells that lack normalgrowth control proliferate in the absence of appropriate growth signals,resulting in damage in structure or function of surrounding tissues.Cells that lack normal differentiation undergo inappropriate phenotypicor physiological changes that do not normally characterize the celltype, resulting in damage in structure and function or surroundingtissues. Cells that lack normal apoptosis fail to undergo, orinappropriately undergo the process of cell death, resulting in damagein structure or function of surrounding tissues. Altered proteinexpression is an example of a phenotype change that renders such cellsdistinguishable from normal. For example, increased or decreasedexpression of a polypeptide normally expressed on a cell, expression ofa mutated polypeptide and expression of a polypeptide not normallyexpressed on a cell are phenotypic changes that can alter a cell fromnormal. Examples of pathologically aberrant cells include tumor cellsand degenerating cells.

As used herein, the term “pathological condition” is intended to mean adisease, abnormal condition or injury of a mammalian cell or tissue.Such pathological conditions include, for example, hyperproliferativeand unregulated neoplastic cell growth, degenerative conditions,inflammatory diseases, autoimmune diseases and infectious diseases.Pathological conditions characterized by excessive or unregulated cellgrowth include, for example, hyperplasia, cancer, autoimmune disease andinfectious disease. Hyperplastic and cancer cells proliferate in anunregulated manner, causing destruction of tissues and organs. Specificexamples of hyperplasias include benign prostatic hyperplasia andendometrial hyperplasia. Specific examples of cancer include prostate,breast, ovary, lung, uterus, brain and skin cancers. Abnormal cellulargrowth can also result from infectious diseases in which foreignorganisms cause excessive growth. For example, human papilloma virusescan cause abnormal growth of skin cells. The growth of cells infected bya pathogen is abnormal due to the alteration of the normal condition ofa cell resulting from the presence of a foreign organism. Specificexamples of infectious diseases include DNA and RNA viral diseases,bacterial diseases, parasitic diseases. Similarly, the growth of cellsmediating autoimmune and inflammatory diseases are aberrantly regulatedwhich results in, for example, the continued proliferation andactivation of immune mechanisms with the destruction of tissues andorgans. Specific examples of autoimmune diseases include, for example,rheumatoid arthritis and systemic lupus erythmatosis. Specific examplesof degenerative disease include osteoarthritis and Alzheimer's disease.

By specific mention of the above categories of pathological conditions,those skilled in the art will understand that such terms include allclasses and types of these pathological conditions. For example, theterm cancer is intended to include all known cancers, whethercharacterized as malignant, benign, soft tissue or solid tumor.Similarly, the terms infectious diseases, degenerative diseases,autoimmune diseases and inflammatory diseases are intended to includeall classes and types of these pathological conditions. Those skilled inthe art will know the various classes and types of proliferative,infectious, autoimmune and inflammatory diseases.

As used herein the term “toll-like receptor 5” or “TLR5” is intended tomean a toll-like receptor 5 of any species, such as the murine and humanpolypeptides containing the amino acid sequences set forth as SEQ IDNOS:6 and 8, respectively, encoded by the nucleic acid sequenceidentified as SEQ ID NOS:5 and 7, respectively. A TLR5 is activated uponbinding to flagellin, an immunomodulatory flagellin peptide, ormodifications thereof, and other TLR5 agonists. Upon activation, a TLR5induces a cellular response by transducing an intracellular signal thatis propagated through a series of signaling molecules from the cellsurface to the nucleus. For example, the intracellular domain of TLR5recruits an adaptor protein, MyD88, which recruits the serine kinaseIRAK. IRAK forms a complex with TRAF6, which then interacts with variousmolecules that participate in transducing the TLR signal. Thesemolecules and other TRL5 signal transduction pathway componentsstimulate the activity of transcription factors, such as fos, jun andNF-kB, and the corresponding induction of gene products of fos-, jun-and NF-kB-regulated genes, such as, for example, TNFa, IL-1 and IL-6.The activities of signaling molecules that mediate the TLR5 signal, aswell as molecules produced as a result of TLR5 activation are TLR5activities that can be observed or measured. Therefore, a TLR5 activityincludes binding to a flagellin polypeptide, immunomodulatory flagellinpeptide, or a modification thereof, recuitment of intracellularsignaling molecules, as well as downstream events resulting from TLR5activation, such as transcription factor activation and production ofimmunomodulatory molecules. A TLR5 cellular response mediates an innateimmune system response in an animal because cytokines released byTLR5-expressing cells regulate other immune system cells to promote animmune response in an animal. Therefore, as used herein the term“TLR5-mediated response” is intended to mean the ability of a flagellinpolypeptide, immunomodulatory peptide or modification thereof to inducea TLR5-mediated cellular response. Exemplary TLR5-mediated cellularresponses include activation of transcription factors such as fos, junand NF-kB, production of cytokines such as IL-1, IL-6 and TNFa, and thestimulation of an immune response in an animal.

A TLR5 also encompasses polypeptides containing minor modifications of anative TLR5, and fragments of a full-length native TLR5, so long as themodified polypeptide or fragment retains one or more biologicalactivities of a native TLR5, such as the abilities to stimulate NF-KBactivity, stimulate the production of cytokines such as TNFa, IL-1, andIL-6 and stimulate an immune response in response to TLR5 binding toflagellin polypeptide, immunomodulatory peptide or modificationsthereof. A modification of a TLR5 can include additions, deletions, orsubstitutions of amino acids, so long as a biological activity of anative TLR5 is retained. For example, a modification can serve to alterthe stability or activity the polypeptide, or to facilitate itspurification. Modifications of polypeptides as described above inreference to flagellin polypeptides and peptides are applicable to TLR5polypeptides of the invention. A “fragment” of a TLR5 is intended tomean a portion of a TLR5 that retains at least about the same activityas a native TLR5.

As used herein, the term “TLR5 agonist” refers to a compound thatselectively activates or increases normal signal transduction throughTLR5. As used herein, the term “TLR5 antagonist” refers to a compoundthat selectively inhibits or decreases normal signal transductionthrough TLR5. A TLR5 agonist or antagonist can alter normal signaltransduction through TLR5 indirectly, for example, by modifying oraltering the native conformation of TLR5 or a TLR5 ligand. Fortherapeutic applications, a TLR5 agonist or antagonist has an EC50 ofless than about 10⁻⁷ M, such as less than 10⁻⁸ M and less than 10⁻⁹ M,although a TRL5 agonist with a higher EC50 can be therapeuticallyuseful. As used herein, the term “TLR5 ligand” refers to a compound thatbinds a TLR5 polypeptide with high affinity. A TLR5 ligand can furtherbe an agonist or antagonist of TLR5, as described above, or can be acompound having little or no effect on TLR5 signaling.

As used herein, the term “detectably labeled” refers to derivitizationwith, or conjugation to, a moiety that is detectable by an analytical orqualitative method. A detectable moiety can be, for example, aradioisotope, such as ¹⁴C, ¹³¹I, ³²P or ³H, fluorochrome, ferromagneticsubstance, or luminescent substance.

As used herein the term “ADCC targeting molecule” is intended to mean anantigen binding protein containing a Fc receptor binding domain capableof inducing antibody-dependent cell cytotoxicity (ADCC). An ADCCtargeting molecule can also contain other domains that augment inductionof ADCC. The flagellin polypeptides and peptides, immunomodulatorypeptides, and modifications described herein, can be domains of an ADCCtargeting molecule that augment induction of ADCC. The ADCC targetingmolecule can include multiple valencies for either or both the antigenbinding domain or the Fc receptor binding domain. Additionally, an ADCCtargeting molecule also can have multiple different antigen bindingdomains combined with a single or multiple copies of an Fc receptorbinding domain or combined with different Fc receptor binding domains.The antigen binding domain or domains can be derived from essentiallyany molecule that has selective or specific binding activity to a targetantigen so long as it can be fused or attached to one or more Fcreceptor binding domains while still maintaining antigen bindingactivity. The Fc receptor binding domain can be derived from an antibodyconstant region of, for example, the IgG class, including subclassesIgG1, IgG3 and IgG4. Such Fc receptor binding domains can be used intheir native form or the amino acid sequence can be modified so as toenhance or optimize the Fc receptor binding or ADCC activity. Moreover,the Fc receptor binding domains can be derived from constant regionswhich recognize either stimulatory or inhibitory Fc receptors. The Fcreceptor binding domain is located within the hinge region of anantibody constant region where the cognate receptors bound by thisdomain include, for example, the Fc RI, Fc RIIA and Fc RIII. Therefore,ADCC targeting molecules include, for example, antibodies selective fora target antigen and functional variants thereof as well as fusionproteins and chemical conjugates containing both an antigen bindingdomain and a Fc receptor binding domain in functionally active forms.ADCC targeting molecules and the use of ADCC targeting molecules in thetreatment of disease are described in detail in U.S. patent applicationSer. No. 09/618,176, which is incorporated herein by reference.

The term “about” when used in reference to a particular activity ormeasurement is intended to refer to the referenced activity ormeasurement as being within a range values encompassing the referencedvalue and within accepted standards of a credible assay within the art,or within accepted statistical variance of a credible assay within theart.

As used herein, the term “substantially” or “substantially the same”when used in reference to an amino acid sequence is intended to meanthat the amino acid sequence shows a considerable degree, amount orextent of sequence identity when compared to the reference sequence.Such considerable degree, amount or extent of identity is furtherconsidered to be significant and meaningful and therefore exhibitcharacteristics which are definitively recognizable or known as beingderived from or related to flagellin. For example, an amino acidsequence which is substantially the same amino acid sequence as anflagellin peptide, including fragments thereof, refers to a sequencewhich exhibits characteristics that are definitively known orrecognizable as being sufficiently related to flagellin so as to fallwithin the classification of flagellin sequences as defined above. Minormodifications thereof are included so long as they are recognizable asan flagellin sequence as defined above.

As used herein, the term “individual” is intended to mean any animal inwhich an immune response can be induced by a flagellin polypeptide,peptide or modifications thereof including a human, non-human primate,cow, pig, chicken, rabbit, ferret, rat or mouse.

An immunomodulatory flagellin polypeptide, peptide or modificationsthereof can be used to induce an immune response in an individual havinga pathological condition, promoting the individual's own immune systemto function more effectively and thereby ameliorate the pathologicalcondition. An individual's immune system may not recognize cancer cellsand other types of pathologically aberrant cells as foreign because theparticular antigens are not different enough from those of normal cellsto cause an immune reaction. In addition, the immune system mayrecognize cancer cells, but induce a response insufficient to destroythe cancer. By stimulating an innate immune response, immunomodulatoryflagellin peptide, polypeptide or modification thereof, promote humoraland cell-mediated responses to antigens on foreign cells orpathologically aberrant cells, such as cancer cells. Administeredindependently or in combination with an antigen, such as a tumorantigen, a flagellin polypeptide, peptide or modification thereof, canbe used to boost the immune system's recognition of cancer cells andother pathologically aberrant cells, and target such cells fordestruction.

Flagellin is a pathogen-associated molecular pattern (PAMP) recognizedby toll-like receptor 5 (TRL5). Toll-like receptor 5 is a member of afamily of at least 10 receptors involved in mediated the innate immuneresponse. Toll-like receptors recognize PAMPs that distinguishinfectious agents from self and mediating the production ofimmunomodulatory molecules, such as cytokines, necessary for thedevelopment of effective adaptive immunity (Aderem, A and Ulevitch, R.J. Nature 406: 782-787 (2000) and Brightbill, H. D., Immunology 101:1-10 (2000)). Members of the toll-like receptor family recognize avariety of antigen types and can discriminate between pathogens. Forexample, TLR2 recognizes various fungal, Gram-positive, andmycobacterial components, TLR4 recognizes the Gram-negative productlipopolysaccharide (LPS), and TLR9 recognizes nucleic acids such as CpGrepeats in bacterial DNA. TLR5 has now been identified as a receptor forbacterial flagellin.

Flagellin induces an innate immune response by binding to and activatingTLR5. Activation of TLR5 by binding to flagellin induces the productionof immunomodulatory molecules, such as cytokines and co-stimulatorymolecules, by a TLR5-expressing cell. For example, activation of TLR5 inmacrophages results in the expression of the cytokines TNFα, IL-1 andIL-6. These cytokines directly and indirectly alter the activities ofimmune system cells that participate in both humoral (TH2) andcell-mediated (TH1) adaptive immune responses. In this manner, animmunomodulatory flagellin peptide, polypeptide or modification thereof,acts as an adjuvant to stimulate a general immune response.

Altering the balance of TH1- versus TH2-associated cytokines can be usedto favorably alter an immune response to treat certain diseases. Forexample, in the use of cancer vaccines, it can be favorable to induceboth TH1 and TH2 responses (Herlyn and Birebent, Ann. Med., 31: 66-78,(1999)). Different sets of cytokines orchestrate TH1 and TH2 immuneresponses. For example, TH1 immune responses are associated with thecytokines IL-2, IFN-g, and TNFα while TH2 immune responses areassociated with the cytokines IL-4, IL-5, IL-6 and IL-10. TLR5stimulates the production of cytokines associated with both TH1- andTH2-associated cytokines. For example, TNFa is associated with thestimulation of a TH1 type immune response (Ahlers, J D et al. J.Immunol, 158: 3947-58 (1997)), and IL-6 is associated with thestimulation of a TH2 type response (Steidler, L. et al. Infect. Immun.,66: 3183-9, (1998)). Therefore, an immunomodulatory flagellin peptide,polypeptide or modification thereof, can be used to advantageouslyelicit TH1 and TH2 type immune responses.

An immunomodulatory flagellin peptide, polypeptide or modificationthereof can also be used to generally alter the particular cytokinesinvolved in an immune response in an individual. Alterations from normallevels of cytokines are observed in many disease states. For thisreason, it can be desirable to increase or decrease the amounts oractivities of specific cytokines involved in particular pathologicalconditions. The cytokines produced in response to TLR5 activation canboth stimulate and down-regulate the production of other cytokines.Therefore, an immunomodulatory flagellin peptide, polypeptide ormodification thereof, or combination of a flagellin molecule with animmunomodulatory molecule or antigen can be used to alter levels ofcytokines associated with a pathological condition. For example, animmunomodulatory flagellin peptide can increase TLR5-expressingmacrophage production of TNFa, IL-1 and IL-6. TNFa and IL-1 generallyfunction as pro-inflammatory cytokines. IL-6 generally functions as ananti-inflammatory cytokine and induces a variety of anti-inflammatoryactivities in immune system cells. For example, IL-6 stimulates theproduction of many anti-inflammatory anti-proteases. Those skilled inthe art will be able to determine if a pathological condition in anindividual could be ameliorated by inducing TLR5-stimulated cytokineproduction and will be able to determine appropriate combinations offlagellin and immunomodulatory molecules suitable for inducing abeneficial immune response.

The invention provides an immunomodulatory flagellin peptide comprisingat least about 10 amino acids of substantially the amino acid sequenceGAVQNRFNSAIT (SEQ ID NO:2), or a modification thereof, that binds totoll-like receptor 5 (TLR5).

The flagellin peptide identified by SEQ ID NO:2 is a peptide of S.Typhimurium1 flagellin which is encoded by the nucleic acid sequenceidentified by SEQ ID NO:1. A flagellin peptide of the invention alsoincludes peptides from other bacterial species, such as H. Pylori (SEQID NO:12), V. Cholera (SEQ ID NO:13), S. marcesens (SEQ ID NO:20), S.flexneri (SEQ ID NO:22), T. Pallidum (SEQ ID NO:23 or SEQ ID NO:24), L.pneumophila (SEQ ID NO:25), B. burgdorferei (SEQ ID NO:26), C. difficile(SEQ ID NO:28), R. meliloti (SEQ ID NO:29), A. tumefaciens (SEQ IDNO:30), R. lupini (SEQ ID NO:31), B. clarridgeiae (SEQ ID NO:33), P.Mirabilis (SEQ ID NO:16), B. subtilus (SEQ ID NO:27), L. monocytogenes(SEQ ID NO:32), P. aeruginosa (SEQ ID NO:14) and E. coli (SEQ ID NO:21),which contain amino acid sequences having 21-71% identity over the 12amino acid sequence of SEQ ID NO:2. A flagellin peptide of the inventionalso includes flagellin peptides from other bacterial species, includingpeptides contained within the flagellin amino acid sequences shown FIG.7. Thus, a flagellin peptide of the invention can have greater thanabout 65% identity, such as greater than about 75%, greater than about85%, greater than about 95%, greater than about 98% identity with thepeptide identified by SEQ ID NO:2.

A flagellin peptide of the invention is derived from a conserved regionof a flagellin polypeptide. Conserved regions of flagellin are wellknown in the art and have been described, for example, inMimori-Kiyosue, et al., J. Mol. Viol. 270: 222-237, (1997). Whereas Tcell receptors which mediate the adaptive immune response recognizerandom portions of antigen amino acid sequences, toll-like receptorsrecognize conserved portions of antigen amino acid sequences. Therefore,the flagellin peptides of the invention and immunomodulatory flagellinpeptides used in the methods of the invention contain amino acidsequences derived from conserved regions of flagellin.

The invention also provides an immunomodulatory flagellin peptidelocated in the D1 region of the flagellin polypeptide. Theimmunomodulatory flagellin peptide includes substantially the samesequence as GALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQ (SEQ ID NO:44), or amodification thereof, and has toll like receptor 5 (TLR5) binding. Thisimmunomodulatory flagellin peptide is described further below in ExampleVII and corresponds to amino acid residues 79-117 of flagellinpolypeptide with reference to the S. Typhimurium 1 amino acid sequence.A schematic of the three dimensional structure of flagellin polypeptideand its corresponding domains D1, D2 and D3 are shown in FIG. 8. FIG. 10shows an alignment of SEQ ID NO:44 with flagellin peptides from thissame amino terminal D1 region identified from other species of flagellinpolypeptides (SEQ ID NOS:51-57).

Other immunomodulatory flagellin peptides located in the amino terminalD1 region of flagellin polypeptide include substantially the same aminoacid sequence TQFSGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTL (SEQ IDNO:45), corresponding to amino acid residues 129-172 of flagellin;EGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVNG (SEQ ID NO:46)corresponding to amino acid residues 78-127 of flagellin orMAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQS (SEQ ID NO:47),corresponding to amino acid residues 0-98 of flagellin, or amodification thereof, where the amino acid residue denoted as “0”corresponds to the initial methonine residue shown in the flagellinsequences in FIG. 7 (and denoted as amino acid residue 1 therein).

The invention also provides and an immunomodulatory flagellin peptidelocated in the D1 region of the flagellin polypeptide and correspondingto the carboxyl terminal portion of a flagellin polypeptide. Theimmunomodulatory flagellin peptide includes substantially the same aminoacid sequence as LQKIDAALAQVDTLRSDLGAVQNRFNSAITNL (SEQ ID NO:48), or amodification thereof, and has toll like receptor 5 (TLR5) binding. Thisimmunomodulatory flagellin peptide is described further below in ExampleVII and corresponds to amino acid residues 408-439 of flagellinpolypeptide with reference to S. Typhimurium 1 amino acid sequence. Aschematic of the three dimensional structure of flagellin polypeptideand its corresponding domains D1, D2 and D3 are shown in FIG. 8. FIG. 10shows an alignment of SEQ ID NO:48 with flagellin peptides from thissame carboxyl terminal D1 region identified from other species offlagellin polypeptides (SEQ ID NOS:58-64).

Other immunomodulatory flagellin peptides located in the carboxylterminal D1 region of flagellin polypeptide include substantially thesame amino acid sequence as TLRSDLGAVQNRFNSAITNLGNTVNNLSS (SEQ IDNO:49), corresponding to amino acid residues 420-448 of flagellin orEQAAKTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLSS (SEQ ID NO:50),corresponding to amino acid residues 398-448 of flagellin, or amodification thereof.

Exemplary modifications of the D1 derived immunomodulatory flagellinpeptides are shown in FIGS. 8-10. Those immunomodulatory flagellinpeptides having modifications that do not substantially affect TLR5binding or TLR5 stimulating activity are included within the modifiedflagellin peptides of the invention.

Also provided are immunomodulatory flagellin peptides containing theabove D1 region flagellin peptides or modifications thereof that areabsent some or all of the flagellin polypeptide D2 and/or D3 regionsequence. Such single chain D1 immunomodulatory flagellin peptidescombine active non-contiguous portions of the amino terminal D1 and thecarboxyl terminal D1 regions into a contiguous peptide sequence. Singlechain D1 immunomodulatory flagellin peptides can be produced by, forexample, deletion of some or all of the D2 or D3 domains oralternatively, the amino terminal D1 region and the carboxyl terminal D1region sequences can be combined using a linker or other moiety thatattaches these domains. Attachment can be through chemical linking ofthe peptides or by recombinant methodology through expression of asingle chain encoding nucleic acid. All of such methods are well knownto those skilled in the art. Single chain D1 immunomodulatory flagellinpeptides can include any one of the amino terminal peptides describedprevious as SEQ ID NOS:44-47 and 51-57 linked in combination with anyone of the carboxyl terminal peptides described previously as SEQ IDNOS:48-50 and 58-65. Immunomodulatory flagellin peptides correspondingto SEQ ID NOS:44-65 derived from other species similarly can be used ina single chain D1 immunomodulatory flagellin peptide of the invention.

As shown in FIGS. 8 and 9, the amino and carboxyl terminal portions of aflagellin polypeptide corresponding to the D1 region associate with eachother by affinity interactions. Similarly, the amino and carboxylterminal components of the single chain D1 immunomodulatory flagellinpeptides of the invention also will associate with each other to mimicthe interactions of these domains in a flagellin polypeptide. Methodsfor constructing active single chain molecules from such interactingdomains are well known to those skilled in the art.

Therefore, the invention provides an immunomodulatory flagellin peptidehaving substantially the same amino acid sequence asGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAE ITQ (SEQ ID NO:44) andsubstantially the same amino acid sequence asLQKIDAALAQVDTLRSDLGAVQNRFNSAITNL (SEQ ID NO:48), or a modificationthereof, and having toll like receptor 5 (TLR5) binding. Single chain D1immunomodulatory flagellin peptides also can have any one ofsubstantially the same amino acid sequence asTQFSGVKVLAQDNTLTIQVGANDGETIDIDLKQINS QTLGLDTL (SEQ ID NO:45);EGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVNG (SEQ ID NO:46) orMAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQS (SEQ ID NO:47), and haveany one of substantially the same amino acid sequence asTLRSDLGAVQNRFNSAITNLG NTVNNLSS (SEQ ID NO:49) orEQAAKTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLSS (SEQ ID NO:50), or amodification thereof.

A flagellin peptide of the invention excludes a portion of flagellindescribed in Newton et al. (supra, 1989), which consists of an S.meunchen flagellin fragment containing a deletion of amino acids207-223, portions of E. coli (strain K12) flagellin described inKuwaijima et al. (supra, 1998), which consist of E. coli flagellinfragments containing deletions of amino acids 239-254, 259-278, 237-262,194-379, 201-318, 218-326, 211-347, 210-299, 245-301, and 220-299, aportion of flagellin described in Samatey et al. (supra, 2000), whichconsists of an S. typhimurium flagellin fragment lacking 52 N-terminalamino acid residues and lacking 44 C-terminal amino acid residues, andportions of flagellin described in McSorley et al. (supra, 2000) whichconsist of S. typhimurium flagellin fragments having the following aminoacid sequences: RSDLGAVQNRFNSAI (SEQ ID NO:40), DLGAVQNRFNSAITN (SEQ IDNO:41), GAVQNRFNSAITNLG (SEQ ID NO:42) AND VQNRFNSAITNLGNT (SEQ IDNO:43).

An immunomodulatory flagellin peptide of the invention can contain aheterologous amino acid sequence that imparts structural or functionalcharacteristics onto the flagellin peptide. For example, chimericflagellin peptides or modifications can be used to impart a targetingfunction. Targeting of a flagellin peptide or modification to aparticular site, such as a mucosal surface for example, confersadditional therapeutic advantage of inducing an immune response at asite of pathological condition or a site favored for inducing anantigen-specific immune response, for example by a vaccine. Further,chimeric flagellin peptides can include a sequence that facilitatesdetection, purification or enhances immunomodulatory activity of theflagellin peptide. A flagellin peptide can be contained, for example, inan ADCC targeting molecule used to treat a pathological condition. Aflagellin peptide can augment the effectiveness of an ADCC targetingmolecule by, for example, stimulating an innate immune response throughTLR5, such as the induction of cytokines such as TNFa, IL-1 and IL-6.Similarly, a flagellin peptide can contain amino acid sequences of avariety of antigen polypeptides, such as those described above inreference to antigens contained in vaccines used in the methods of theinvention. A chimeric flagellin peptide containing amino acid sequencesof an antigen or containing an antigenic molecule such as acarbohydrate, nucleic acid, or lipid, can be used analogously to avaccine, as described above, as well as in a vaccine formulation, toinduce an immune response in an individual. As such, a chimericflagellin peptide can be a vaccine that induces both innate and adaptiveimmune system responses.

An immunomodulatory flagellin peptide of the invention can be preparedby a variety of methods well-known in the art, for example, byrecombinant expression systems described below, and biochemicalpurification methods described below, as well as by synthetic methodswell known in the art. Methods for recombinant expression andpurification of polypeptides in various host organisms are described,for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, New York (1992) and in Ansubel et al.,Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore,Md. (1998), both of which are incorporated herein by reference.Similarly, flagellin peptide modifications can be generated usingrecombinant mutagenesis, such as site directed mutagenesis and PCRmutagenesis, and expression of the flagellin peptide modification.Numerous methods of constructing, modifying, expressing and purifyingpeptides are known to those skilled in the art. A specific example of amethod for purifying flagellin is described below in Example III. Thechoice of recombinant methods, expression and purification systems willbe known by those skilled in the art and will depend on the user and theparticular application for the immunomodulatory flagellin peptide ormodification thereof.

A flagellin peptide of the invention induces an innate immune responsein an individual by binding to an stimulating TLR5. Therefore, theinvention provides methods for inducing an immune response in anindividual having a pathological condition that can be ameliorated byimmune system activity. The methods involve administering animmunomodulatory flagellin peptide or modification thereof to induce animmune response, administering a combination of an immunomodulatoryflagellin peptide and an antigen to induce an antigen-specific immuneresponse, and administering a combination of an immunomodulatoryflagellin peptide and an immunomodulatory molecule to modulate an immuneresponse. The selection of a particular method for inducing an immuneresponse will depend on the particular pathological condition to beameliorated or prevented in an individual. As described herein, themethods are applicable to a wide variety of pathological conditions.Those skilled in the art will be able to determine if an immune responsecan be beneficially modulated by administering an immunomodulatoryflagellin peptide or combination thereof with an antigen orimmunomodulatory molecule.

The invention provides method of inducing an antigen-specific immuneresponse in an individual. The method involves administering to anindividual an immunogenic amount of a vaccine, comprising an antigen andan immunomodulatory flagellin peptide having at least about 10 aminoacids of substantially the amino acid sequence of SEQ ID NO:2, or amodification thereof.

As an adjuvant in a vaccine formulation, the immunomodulatory flagellinpeptides of the invention can contribute to the effectiveness of thevaccine by, for example, enhancing the immunogenicity of weaker antigenssuch as highly purified or recombinant antigens, reducing the amount ofantigen required for an immune response, reducing the frequency ofimmunization required to provide protective immunity, improve theefficacy of vaccines in individuals with reduced or weakened immuneresponses, such as newborns, the aged, and immunocompromisedindividuals, and enhance the immunity at a target tissue, such asmucosal immunity, or promote cell-mediated or humoral immunity byeliciting a particular cytokine profile. An immunomodulatory flagellinpeptide, polypeptide or modification thereof induces an innate immuneresponse through activation of TLR5. The innate immune responseincreases the immune response to an antigen by stimulating the adaptiveimmune response. Therefore, a combination of an immunomodulatoryflagellin peptide, polypeptide or modification thereof with one or moreantigens provides an effective vaccine for inducing an immune responsein an individual.

The methods of the invention for inducing an antigen-specific immuneresponse can be used to treat individuals having a variety ofpathological conditions. For example, cancer vaccines have been usedeffectively for treating melanoma and breast cancers. Vaccines have beenused for treatment of inflammatory diseases such as asthma (Scanga C. Band Le Gros, G., Drugs 59(6), 1217-1221 (2000)), infectious diseases ofpathogenic bacteria such as H. pylori, pathogenic viruses such as humanpapilloma virus and HIV (Sutton P. and Lee, A, Aliment Pharmacol. 14:1107-1118 (2000)), protozoa, autoimmune diseases such as diabetes (vonHerrath and Whitton, Ann. Med. 32: 285-292 (2000)) and degenerativediseases such as Alzheimer's disease (Youngkin, S. G., Nat. Med., 7(1):18-19 (2001)). Therefore, a vaccine used in the methods of the inventionfor inducing an antigen-specific immune response can be administered toan individual for treatment of a variety of pathological conditions,including proliferative disease, infectious disease, inflammatorydisease and degenerative disease.

A variety of antigens can be used in combination with animmunomodulatory flagellin peptide of the invention for preparing avaccine. Microorganisms such as viruses, bacteria and parasites containsubstances that are not normally present in the body. These substancescan be used as antigens to produce an immune response to destroy boththe antigen and cells containing the antigen, such as a bacterial cellor cancer cell.

For example, isolated or crude antigens of microbial pathogens can beused in vaccines to treat infectious disease; isolated or crude tumorcell antigens can be used in vaccines to treat cancer; isolated or crudeantigens known to be associated with a pathologically aberrant cell canbe used to treat a variety of diseases in which it is beneficial totarget particular cells for destruction.

A variety of substances can be used as antigens in a vaccine compound orformulation. For example, attenuated and inactivated viral and bacterialpathogens, purified macromolecules, polysaccharides, toxoids,recombinant antigens, organisms containing a foreign gene from apathogen, synthetic peptides, polynucleic acids, antibodies and tumorcells can be used to prepare a vaccine useful for treating apathological condition. Therefore, an immunomodulatory flagellin peptideof the invention can be combined with a wide variety of antigens toproduce a vaccine useful for inducing an immune response in anindividual. Those skilled in the art will be able to select an antigenappropriate for treating a particular pathological condition and willknow how to determine whether a crude or isolated antigen is favored ina particular vaccine formulation.

An isolated antigen can be prepared using a variety of methods wellknown in the art. A gene encoding any immunogenic polypeptide can beisolated and cloned, for example, in bacterial, yeast, insect, reptileor mammalian cells using recombinant methods well known in the art anddescribed, for example in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1992) and inAnsubel et al., Current Protocols in Molecular Biology, John Wiley andSons, Baltimore, Md. (1998). A number of genes encoding surface antigensfrom viral, bacterial and protozoan pathogens have been successfullycloned, expressed and used as antigens for vaccine development. Forexample, the major surface antigen of hepatitis B virus, HbsAg, the bsubunit of choleratoxin, the enterotoxin of E. coli, thecircumsporozoite protein of the malaria parasite, and a glycoproteinmembrane antigen from Epstein-Barr virus, as well as tumor cellantigens, have been expressed in various well known vector/host systems,purified and used in vaccines. An immunomodulatory flagellin peptide,polypeptide or modification thereof induces an innate immune responsethrough TLR5 that can beneficially enhance an immune response to arecombinant antigen.

A pathologically aberrant cell to be used in a vaccine can be obtainedfrom any source such as one or more individuals having a pathologicalcondition or ex vivo or in vitro cultured cells obtained from one ormore such individuals, including a specific individual to be treatedwith the resulting vaccine.

Those skilled in the art will be able to determine if a vaccine compoundor formulation induces an innate, humoral, cell-mediated, or anycombination of these types of immune response, as methods forcharacterizing these immune responses are well known in the art. Forexample, the ability of a vaccine compound or formulation to induce aninnate immune response through TLR5 can be determined using methodsdescribed herein as well as other methods. Such methods for detecting aninnate immune response can be generally performed within hours ofvaccine administration. The ability of a vaccine compound or formulationto induce a humoral response can be determined by measuring the titer ofantigen-specific antibodies in an animal primed with the vaccine andboosted with the antigen, or determining the presence of antibodiescross-reactive with an antigen by ELISA, Western blotting or otherwell-known methods. Cell-mediated immune responses can be determined,for example, by measuring cytotoxic T cell response to antigen using avariety of methods well known in the art. Methods of detecting humoraland cell-medicated immune responses can be generally performed days orweeks after vaccine administration.

A combination of an antigen or immunomodulatory molecule and animmunomodulatory flagellin peptide, polypeptide or modification thereof,can be tested in a variety of preclinical toxicological and safetystudies well known in the art. For example, such a combination can beevaluated in an animal model in which the antigen has been found to beimmunogenic and that can be reproducibly immunized by the same routeproposed for human clinical testing. A combination of an antigen orimmunomodulatory molecule and an immunomodulatory flagellin peptide ormodification thereof can be tested, for example, by an approach setforth by the Center for Biologics Evaluation and Research/Food and DrugAdministration and National Institute of Allergy and Infectious Diseases(Goldenthal, K L et al. AID Res Hum Retroviruses, 9: S45-9 (1993)).

Those skilled in the art will know how to determine for a particularcombination of antigen or immunomodulatory molecule and immunomodulatoryflagellin polypeptide modification thereof, the appropriate antigenpayload, route of immunization, volume of dose, purity of antigen, andvaccination regimen useful to treat a particular pathological conditionin a particular animal species.

The invention provides a method of inducing a TLR5-mediated response.The method involves administering to a TLR5-contain cell an effectiveamount of an immunomodulatory flagellin peptide having at least about 10amino acids of substantially the amino acid sequence of SEQ ID NO:2, ora modification thereof.

A TLR5-mediated response can be assessed in a cell or animal becauseTLR5 stimulates cellular activities that stimulate the immune responsethat occurs in an animal. For example, flagellin binding to TLR5 inducescellular events such as an increase in the amount or activity ofcytokines, such as TNFa, IL-1 and IL-6. These cytokines in turn regulatethe activities of immune system cells. Therefore a TLR5-mediatedresponse can be determined by examining an immune responses in an animaland by observing particular immune system cell activities. Determinationof immune responses in an animal is discussed below. Determination ofimmune system cell activities can be performed, for example, byobserving or measuring the amount of activity of immunomodulatorymolecules produced by specific types of immune cells. Cytokineproduction by macrophages is an exemplary immune cell activity that canbe conveniently measured using methods well known in the art and thosedescribed herein. A biological activity of a cytokine can also beassessed using methods well known in the art. TNFa activities include,for example, inducing the production of IL-1 and IL-6, activation ofneutrophils and endothelial cells in inflammation, inducing acute phasereactants in liver, inducing fever. IL-1 activities include, forexample, activating of endothelial cells in inflammation andcoagulation, inducing acute phage reactants in liver, inducing fever andstimulating T cell proliferation. IL-6 activities include, for example,stimulating proliferation of mature B cells and inducing their finalmaturation into antibody-producing plasma cells, inducing IL-2 receptorexpression, inducing acute phase reactants in liver, and co-stimulationof thymocytes in vitro. A regulatory effect of IL-6 is inhibition ofTNFa production, providing negative feedback for limiting the acuteinflammatory response (Feghali, C. A. and Wright, T. M., Frontiers inBioscience, 2, d12-26 (1997) provides a summary of cytokine activities).

The invention provides a method of inducing an immune response in anindividual having a pathological condition. The method involvesadministering to said individual an immunogenic amount of animmunomodulatory flagellin peptide having at least about 10 amino acidsof substantially the amino acid sequence of SEQ ID NO:2, or amodification thereof.

As described above, an immunomodulatory flagellin peptide can be used tobeneficially boost a general immune response in an individual having apathological condition by stimulating an innate immune response. Anincreased immune response can ameliorate a pathological condition aswell as prevent a pathological condition in a healthy individual, orindividual not having a pathological condition. Therefore, animmunomodulatory flagellin peptide can be administered prophylacticallyto an individual not having a pathological condition, if desired.

The invention provides another method of modulating an immune responsein an individual having a pathological condition. The method involvesadministering to the individual a combination of an immunogenic amountof an immunomodulatory flagellin peptide having at least about 10 aminoacids of substantially the amino acid sequence of SEQ ID NO:2, or amodification thereof, and another immunomodulatory molecule.

As described above, a combination of an immunomodulatory flagellinpeptide with another immunomodulatory molecule can be used toadvantageously induce or modulate an immune response. An immune responsecan be induced by combining an immunomodulatory flagellin peptide withanother immunomodulatory molecule that induces an immune response in ageneral manner, such as an adjuvant, or can be combined with animmunomodulatory molecule that induces a particular alteration in animmune cell activity. Such immunomodulatory molecules are describedherein.

Modulating an immune response is useful for promoting a more effectiveor more normal immune response in an individual having a pathologicalcondition. As described above, alterations in normal cytokine levels areassociated with various pathological conditions. An immunomodulatoryflagellin peptide or combination with another immunomodulatory moleculecan be used to modulate cytokine levels in an individual by inducing theproduction of immunomodulatory molecules, such as cytokines includingTNFα, IL-1, and IL-6 through TLR5, and inducing the production ofsuppression of the same or different immunomodulatory molecules throughthe activity of the administered immunomodulatory molecule. Therefore,the immunomodulatory flagellin peptides of the invention can be combinedwith immunomodulatory molecules that alter an immune response bystimulating or inhibiting the cellular functions of immune system cells.

A variety of immunomodulatory molecules can be used in combination withan immunomodulatory flagellin peptide or modification thereof of theinvention to alter an immune response in an individual. The type ofalteration desired will determine the type of immunomodulatory moleculeselected to be combined with an immunomodulatory flagellin peptide. Forexample, to promote an innate immune response, a immunomodulatoryflagellin peptide can be combined with another immunomodulatory moleculethat promotes an innate immune response, such as a PAMP or conservedregion known or suspected of inducing an innate immune response. Avariety of PAMPs are known to stimulate the activities of differentmembers of the toll-like family of receptors. Such PAMPs can be combinedto stimulate a particular combination of toll-like receptors that inducea beneficial cytokine profile. For example, PAMPs can be combined tostimulate a cytokine profile that induces a TH1 or TH2 immune response.

Other types of immunomodulatory molecules that promote humoral orcell-mediated immune responses can be combined with a flagellin moleculeof the invention. For example, cytokines can be administered to alterthe balance of TH1 and TH2 immune responses. Those skilled in the artwill know how to determine the appropriate cytokines useful forobtaining a beneficial alteration in immune response for a particularpathological condition.

Immunomodulatory molecules that target antigens and cells displayingantigens for destruction can be combined with a flagellin molecule ofthe invention. For example, the effectiveness of monoclonal antibodiesand ADCC targeting molecules that recognize a particular antigen on anunwanted cell, such as a pathologically aberrant cell can be increasedwhen administered with a flagellin molecule of the invention.Immunomodulatory molecules that stimulate or suppress cellularactivities such as proliferation, migration, activation, interaction anddifferentiation can be combined with a flagellin molecule of theinvention. For example, IL-2 can be used to stimulate proliferation ofimmune system cells, certain interferons can be used to interfere withthe rapid growth of cancer cells or to interfere with angiogenesis, andganulocyte-colony stimulating factor can be used to increase productionof certain types of immune system cells and blood cells. A variety ofimmunostimulating and immunosupressing molecules and modalities are wellknown in the art and can be used in combination with a flagellinpolypeptide, peptide or modification thereof, of the invention. Aflagellin molecule of the invention increases the beneficial effect ofan immunomodulatory molecule by inducing TLR5-mediated production ofimmunomodulatory molecules that function in concert with a selectedimmunomodulatory molecule to produce a desired cytokine profile orcellular activity, or prime the adaptive immune response to respond tothe selected immunomodulatory molecule.

The methods of the invention for using immunomodulatory flagellinpeptides to induce an immune response are also applicable to a flagellinpolypeptide, or a modification thereof. Accordingly, the inventionprovides a method of inducing an immune response in an individual,including a human, having a pathological condition. The method involvesadministering to the individual an immunogenic amount of animmunomodulatory flagellin polypeptide, or modification thereof, whenthe flagellin polypeptide induces an immune response.

An immunomodulatory flagellin peptide of the invention binds to TLR5,and stimulates a TLR5 activity. The ability of an immunomodulatoryflagellin peptide or modification thereof to bind to TLR5 or stimulate aTLR5 activity can be determined using methods known in the art. Methodsof determining specific binding interactions of flagellin peptides andmodifications thereof with TLR5 can be determined using well knownmethods in the art such as methods of trapping ligand-receptor complexesusing chemical cross-linking, and competitive inhibition of reagentsspecific for TLR5 such as specific flagellin peptides or modifications,antibodies or other TLR-5 specific reagents.

Methods of determining TLR5 functional activities in response to animmunomodulatory flagellin peptide or modification thereof includemethods described herein, in Examples I through IV, as well as methodsknown in the art. A variety of methods well known in the art can be usedfor determining transcription factor activities. For example, fos, jun,and NF-kB activation in response to TLR5 binding to a flagellin moleculecan be detected by electrophoretic mobility shift assays well known inthe art that detect NF-kB binding to specific polynucleic acidsequences, and promoter-reporter nucleic acid constructs such that, forexample, b-lactamase, luciferase, green fluorescent protein orb-galactosidase will be expressed in response to contacting a TLR5 witha flagellin polypeptide, peptide or equivalent thereof. For example, aluciferase reporter plasmid in which luciferase protein expression isdriven by one or more NF-kB binding sites can be transfected into acell, as described in Examples I-IV. Activation of NF-KB results inactivation of luciferase reporter expression, resulting in production ofluciferase enzyme able to catalyze the generation of a molecule that canbe detected by colorimetric, fluorescence, chemilluminescence orradiometric assay.

An amount or activity of a polypeptide, including a cytokine such asTNFa, IL-1 or IL-6, can be a read-out for activation of a TLR5 inresponse to binding an immunomodulatory flagellin peptide ormodification thereof. A variety of methods well known in the art can beused to measure cytokine amounts, such as, for example, flow cytometrymethods, immunoassays such as ELISA and RIA, and cytokine RNA protectionassays. Commercially available cytokine assay kits, such as ELISA assayformats, can be conveniently used to determine the amount of a varietyof cytokines in a sample. Those skilled in the art will determine theparticular cytokines to be measured when assessing an immune response ina cell or animal. For example, to determine whether a particularresponse is characterized as a TH1 or TH2 immune response, those skilledin the art will be able to select appropriate cytokines within the TH1and TH2 categories, which are well known in the art.

A sample used for determining a TLR5-mediated response or immuneresponse can include, for example, a fluid or tissue obtained from ananimal, a cell obtained from an animal fluid or tissue, cultured cellsincluding in vitro and ex vivo cultured cells, and lysates or fractionsthereof and cultured cells that express TLR5.

An immune response in an animal is determined by the collectiveresponses of the cells of the immune system. An immune response can bedetected by observing various indicators of immune response in ananimal. Such indicators include, for example, visible signs ofinflammation of tissues, such as swelling, production of antibodies,such as levels of IgA, IgG and IgM in blood and levels of IgA in saliva,alterations in immune cell numbers, such as increased or decreasedproliferation of particular immune cells, and in immune cell activities,such as production of immunomodulatory molecules and second messengermolecules. For example, an immune response to a particular antigen canbe observed in a animal using methods well known in the art such asdelayed hypersensitivity skin tests. An immune response can bedetermined by the presence of antibodies cross reactive with an antigen,such as by ELISA and Western blotting, lymphocyte activation testsemploying mitogen or antigen stimulation, mixed lymphocyte culturetests, assays for human T and B lymphocytes, flow cytometry and cellsorting to characterize populations of immune system cells obtained froman individual, soluble antigen uptake by macrophages, and tests ofneutrophil functions (Stites et al. Basic and Clinical Immunology,4^(th) edition, Lange Medical Publications, Los Altos, Calif. (1982)).An immune response can also be assessed by examining amounts oractivities of immune system mediators, such as cytokines and chemokines,in cells collected from fluids or tissues of animals. A variety ofmethods are well known in the art for qualitative and quantitativemeasurement of cytokine amount and bioassay of cytokine activity.

The methods of the invention for inducing an immune response can be usedto treat any animal species having an immune response upon treatmentwith flagellin polypeptide, peptide, or modification thereof, and forwhich a stimulation of an immune response is desired. Such animalsinclude avian species such as chicken, and mammalian species such asrodent, canine, feline, bovine, porcine and human subjects. Methods forusing adjuvants with vaccines and vaccinating animals are well known inthe art and are routinely used in laboratory animals. Those skilled inthe art will be able to determine if a particular animal species has aflagellin-stimulated TLR5-mediated innate immune response.

A vaccine to be used in the methods of the invention for inducing animmune response can be administered as a solution or suspension togetherwith a pharmaceutically acceptable medium. Such a pharmaceuticallyacceptable medium can be, for example, water, phosphate buffered saline,normal saline or other physiologically buffered saline, or other solventor vehicle such as glycol, glycerol, and oil such as olive oil or aninjectable organic ester. A pharmaceutically acceptable medium can alsocontain liposomes or micelles, and can contain immunostimulatingcomplexes prepared by mixing polypeptide or peptide antigens withdetergent and a glycoside, such as Quil A. Further methods for preparingand administering an immunomodulatory flagellin polypeptide or peptide,or modification in a pharmaceutically acceptable medium are presentedbelow, in reference to compounds that induce a TLR-mediated response.

The immunomodulatory flagellin polypeptides, peptides and modificationsthereof used in the methods of the invention can be administered by avariety of routes to stimulate an immune response. For example, theseimmunomodulatory molecules can be delivered intranasally,subcutaneously, intradermally, intralymphatically, intra-muscularly,intratumorally, orally, intravesically, intraperitoneally andintracerebrally. Oral administration is convenient and relatively safe.Oral vaccination protocols can be useful for inducing the state ofimmunological tolerance which normally occurs in response to mostsoluble antigens and the proteolytic degradation of antigen preparationsin the digestive tract. Nasal delivery routes may be useful for inducingboth mucosal and systemic immune responses. A variety of devices areunder development for convenient and effective delivery of formulationsto the nasal cavity and pulmonary tissues. Those skilled in the art willknow how to select appropriate delivery routes for particularformulations of flagellin polypeptides, peptides and modificationsthereof.

The invention provides a screening composition consisting of animmunomodulatory flagellin peptide comprising at least about 10 aminoacids of substantially the amino acid sequence GAVQNRFNSAIT (SEQ IDNO:2), or a modification thereof, and having toll-like receptor 5 (TLR5)binding, and a TLR5. The composition is useful for identifying agonists,antagonists and ligands for TLR5. The characteristics of animmunomodulatory flagellin peptide comprising at least about 10 aminoacids of substantially the amino acid sequence GAVQNRFNSAIT (SEQ IDNO:2), or a modification thereof, and having toll-like receptor 5 (TLR5)binding, and preparation of a flagellin peptide are described herein.Similarly, the characteristics of a TLR5 polypeptide and modificationsthereof that have a TLR5 activity, and methods for preparing a TLR5polypeptide to be used in the methods of the invention are describedherein. Chimeric TLR5s, such as the CD4-TLR5 described herein in ExampleI, are included in the screening compositions of the invention.

The screening composition of the invention includes, for example, cells,cell extracts and artificial signaling systems that contain a TLR5polypeptide or modification thereof. The cell compositions of theinvention include any cell in which TLR5 can couple to a signaltransduction pathway to produce a detectable signal in response to anagonist, such as flagellin or a flagellin peptide. Such cells includeinsect cells such as Drosophila cells, yeast cells such as S.cerevisiae, prokaryotic cells such as E. coli, amphibian cells such asXenopus oocytes, and vertebrate cells such as mammalian primary cells,such as macrophages. Primary cells such as macrophages and otherlymphocytes can be conveniently isolated from blood using methods wellknown in the art. Cells obtained from transgenic animals, such astransgenic mice that have been engineered by known methods of expressrecombinant TLR5 or TLR5 signal transduction components are alsoincluded in the screening compositions of the invention. Cell linesprepared from any of theses cell types, such as S2, CHO, NIH-3T3, 293and HeLa cells are also included in a screening composition of theinvention.

The screening compositions of the invention can include crude orpartially purified lysates or extracts of the cell compositions of theinvention, and reconstituted signaling systems. Artificial signalingsystems include, for example, natural or artificial lipid bilayers, suchas a liposome or micelle, which promote an active conformation of aTLR5. The compositions can further contain cellular fractions orisolated components necessary for producing and detecting the desiredpredetermined signal.

The invention provides a method of screening for a TLR5 ligand, agonistor antagonist. The method involves, (a) contacting a TLR5 with acandidate compound in the presence of a flagellin polypeptide orimmunomodulatory flagellin peptide under conditions wherein binding ofthe flagellin polypeptide or immunomodulatory flagellin peptide to theTLR5 produces a predetermined signal; (b) determining the production ofthe predetermined signal in the presence of the candidate compound; and(c) comparing the predetermined signal in the presence of the candidatecompound with a predetermined signal in the absence of the candidatecompound, wherein a difference between the predetermined signals in thepresence and absence of the candidate compound indicates that thecompound is a TLR5 ligand, agonist or antagonist.

TLR5 can produce a variety of predetermined signals useful in themethods of the invention for identifying a TLR5 ligand, agonist orantagonist. TLR5 has an extracellular domain that participates in ligandrecognition and intracellular domain that contain a conserved regioncalled the Toll/IL-IR homology (TIR) domain that, upon activation,recruits an adaptor protein, MyD88. Through an amino terminal deathdomain, MyD88 recruits the serine kinase IRAK to propagate apro-inflammatory signal through binding to TRAF6, which then binds toother molecules that participate in the TLR5 signaling cascade.Immunomodulatory flagellin peptides and modifications binding to TLR5induces signal transduction events which result in, for example,stimulating NF-kB activity and inducing production of gene products ofNF-kB-regulated genes, such as TNFa, IL-1 and IL-6, as well asstimulating AP-1 transcription factors fos and jun. Therefore, apredetermined signal can include a signal produced by animmunomodulatory flagellin polypeptide or peptide or modificationbinding to TLR5, a signal produced by a TLR5 intracellular signaltransduction even, such as kinase or phosphatase activity orprotein-protein interactions, by activation of fos, jun or NF-kB, and byan amount or activity of a fos-, jun- or NF-kB-regulated gene or geneproduct, such as TNFa, IL-1 and IL-6.

A variety of low- and high-throughput assays suitable for detectingselective binding interactions between a receptor and a ligand are knownin the art. Both direct and competitive assays can be performed,including, for example, fluorescence correlation spectroscopy (FCS) andscintillation proximity assays (SAP) reviewed in Major, J. Receptor andSignal Transduction Res. 15: 595-607 (1995); and in Sterrer et al., J.Receptor and Signal Transduction Res. 17: 511-520 (1997)). Other assaysfor detecting binding interactions include, for example, ELISA assays,FACS analysis, and affinity separation methods. Such assays can involvelabeling a TLR5 ligand, such as flagellin or a flagellin peptide, with adetectable moiety such as a radiolabel, fluorochrome, ferromagneticsubstance, or luminescent substance. A detectably labeled flagellinpolypeptide or peptide can be prepared using methods well known in theart. Receptor binding assays, including high-throughput automatedbinding assays, and methods of determining binding affinity from suchassays, are well known in the art, and any suitable direct orcompetitive binding assay can be used. Exemplary high-throughputreceptor binding assays are described, for example, inMellentin-Micelotti et al., Anal. Biochem. 272: P182-190 (1999); Zuck etal., Proc. Natl. Acad. Sci. USA 96: 11122-11127 (1999); and Zhang etal., Anal. Biochem. 268; 134-142 (1999).

A variety of methods well known in the art can be used to detectactivation of transcription factors, such as NF-kB, in low- orhigh-throughput formats. The methods described herein and in theExamples can be adapted to formats suitable for candidate compoundscreening.

A variety of low- and high-throughput assays suitable for detectingamounts and activities of polypeptides such as cytokines are known inthe art. Methods for detecting polypeptides, include, for example, flowcytometric measurements as described herein, immunodetection methodssuch as radioimmune assay (RIA), ELISA, immunoprecipitation and Westernblotting. Assay of the activity of a cytokine include function bioassaysand detection of amounts of polypeptides regulated by a particularcytokine. Those skilled in the art can determine an appropriate methodfor detecting an activity of a particular cytokine.

Suitable conditions under which TLR5 produces a predetermined signal inresponse to a flagellin polypeptide, peptide or modification can bedetermined by those skilled in the art, and will depend on theparticular predetermined signal selected. Exemplary conditions fordetermining the production of a predetermined signal are provided hereinin Examples I-IV. Any known or predicted TLR5-mediated cellular event,such as elicitation of second messengers, induction of gene expressionor altered cellular proliferation, differentiation or viability can be apredetermined signal that is an indication of activation of signaltransduction through TLR5.

Assays for detecting a predetermined signal produced by binding offlagellin or flagellin peptide to TLR5 can be performed, for example,with whole cells that express TLR5, membrane fractions, or artificialsystems, as described herein, or with isolated TLR5 polypeptide, eitherin solution, in an artificial membrane, or bound to a solid support.

A method of identifying TLR5 agonists and antagonists can be performedeither in the presence of a predetermined concentration of a known TLR5agonist, such as flagellin, flagellin peptide, or modifications thereof,or in the absence of agonist. The agonist can be added either prior to,simultaneously with, or after, addition of the test compound. Whenpresent, the agonist concentration is preferably within 10-fold of itsEC50 under the assay conditions to allow the identification of acompound that competes with a known agonist for signaling through TLR5,or indirectly augments signaling through the receptor. Likewise, acompound that reduces binding between a known agonist and its receptor,or indirectly decreases signaling through the receptor, can also beidentified.

The method of screening to identify a ligand, agonist or antagonist ofTLR5 involve testing a candidate compound. A candidate compound can beany substance, molecule, compound, mixture of molecules or compounds, orany other composition. The candidate compounds can be small molecules ormacromolecules, such as biological polymers, including proteins,polysaccharides and nucleic acids. Sources of candidate compounds whichcan be screened for a ligand, agonist or antagonist of TLR5 include, forexample, libraries of small molecules, peptides and polypeptides.

Additionally, candidate compounds can be preselected based on a varietyof criteria. For example, suitable candidate compounds can be selectedas having known ligand, agonist or antagonist activity. Alternatively,candidate compounds can be selected randomly. Candidate compounds can beadministered to the reaction system at a single concentration or,alternatively, at a range of concentrations to determine, for example,an EC50 or IC50 of a candidate compound.

The method of screening for TLR5 ligands, agonists or antagonists caninvolve groups or libraries of compounds. Methods for preparing largelibraries of compounds, including simple or complex organic molecules,carbohydrates, peptides, peptidomimetics, polypeptides, nucleic acids,antibodies, and the like, are well known in the art. Librariescontaining large numbers of natural and synthetic compounds can beobtained from commercial sources.

The number of different candidate compounds to examine using the methodsof the invention will depend on the application of the method. It isgenerally understood that the larger the number of candidate compounds,the greater the likelihood of identifying a compound having the desiredactivity in a screening assay. Large numbers of compounds can beprocessed in a high-throughput automated format.

The TLR5 agonists, antagonists and ligands identified using the methodsand compositions described herein, are potential therapeutic compoundsthat can be administered to an individual, such as a human or othermammal, in an effective amount to increase or decrease signaling throughTLR5, for example, to alter an immune response or treat aTLR5-associated condition. Such compounds can be used analogously toimmunomodulatory compounds useful for augmenting and altering an immuneresponse, as described above. For example, a compound can be used toinduce a general immune response and to induce a specific immuneresponse in the presence of an antigen and to alter the level of aparticular cytokine in an individual having a pathological condition.

The TLR5 agonists and antagonists, immunomodulatory flagellin peptides,polypeptides and modifications thereof, are useful for ameliorating, orreducing the severity of a pathological condition. Reduction in severityincludes, for example, an arrest or decrease in clinical symptoms,physiological indicators, biochemical markers or metabolic indicators ofdisease. Those skilled in the art will know, or will be able todetermine the appropriate clinical symptoms, physiological indicators,biochemical markers or metabolic indicators to observe for a particularpathological condition. To prevent a disease means to preclude theoccurrence of a disease or restoring a diseased individual to theirstate of health prior to disease.

In addition to applications described herein for agonists andantagonists, a TLR5 ligand can be used, for example, to specificallytarget a diagnostic moiety to cells and tissues that express TLR5, suchas monocytes, immature dendritic cells, epithelial cells, and othercells involved in an immune response. Thus, a TLR5 ligand can be labeledwith a detectable moiety, such as a radiolabel, fluorochrome,ferromagnetic substance, or luminescent substance, and used to detectnormal or abnormal expression of TLR5 polypeptide in an isolated sampleor in vivo diagnostic imaging procedures.

A heterologous amino acid sequence can be advantageously used to providea tag for detection or purification or to impart an activity to areference polypeptide or peptide, such as an enzyme activity, abiological activity, an immunological activity or stability. Animmunomodulatory flagellin peptide, polypeptide or modification thereof,or TLR5 polypeptide can contain a heterologous amino acid sequence, oramino acid sequence not present in the native amino acid sequence of areference polypeptide or peptide and not represented by a modificationof a reference polypeptide or peptide. A heterologous amino acidsequence can be of any size in relation to the reference amino acidsequence. A TLR5 polypeptide containing the heterologous sequence of CD4is a specific example of such a modification and is described further inExample I. The described CD4-TLR5 chimera is identified by the aminoacid sequence of SEQ ID NO:10, encoded by the nucleic acid sequence ofSEQ ID NO:9. A chimeric TLR5 can be prepared using cloning methods wellknown in the art. For example, a chimeric polypeptide can be produced byamplifying by PCR a nucleotide sequence encoding a portion of a selectedpolypeptide using sequence specific primers. Primers useful foramplifying a TLR5 include, for example, huTLR5-A6:TTAAAGTGGTACCAGTTCTCCCTTTTCATTGTATGCACT (SEQ ID NO:35) and huTLR5DNS:CGGGATCCCGTTAGGAGATGGTTGCTACAGTTTGC (SEQ ID NO:36). A portion of a TLR5nucleotide sequence, such as a sequence amplified using such primers canbe fused to a nucleotide sequence encoding a heterologous amino acidsequence. A variety of methods for generating nucleic acid sequencesencoding chimeric polypeptides are well known to those skilled in theart.

The polypeptides and peptides described herein, includingimmunomodulatory flagellin peptides, flagellin polypeptide, TLR5polypeptides and fragments thereof can be prepared using a variety ofprotein expression systems well known in the art, including prokaryoticand eukaryotic expression systems. Prokaryotic expression systems areadvantageous due to their ease in manipulation, low complexity growthmedia, low cost of growth media, rapid growth rates and relatively highyields. Well known prokaryotic expression systems include, for example,E. coli bacterial expression systems based on bacteriophage T7 RNApolymerase, the trc promoter, the araB promoter and bacillus expression.Eukaryotic expression systems are advantageous because expressedpolypeptides can contain eukaryotic post-translational modificationssuch as O-linked glycosylation, phosphorylation and acetylation and canhave improved protein folding. Well known eukaryotic expression systemsinclude, for example, expression in yeast, such as Pichia pastoris andPichia methanolica, expression in insect systems such as the DrosophilaS2 system and baculovirus expression systems and expression in mammaliancells using adenoviral vectors and cytomegalovirus promoter-containingvectors.

An immunomodulatory flagellin peptide, polypeptide, TLR5 or fragmentsthereof can be purified using a variety of methods of proteinpurification well known in the art. Biochemical purification caninclude, for example, steps such as solubilization of the polypeptide orpeptide-expressing cell, isolation of the desired subcellular fractions,chromatography, such as ion exchange, size, or affinity-basedchromatographies, electrophoresis, and immunoaffinity procedures. Otherwell-known methods are described in Deutscher et al., Guide to ProteinPurification: Methods in Enzymology Vol. 182, (Academic Press, (1990)).An exemplary method for purifying a flagellin peptide is provided inExample III. The methods and conditions for biochemical purification ofa polypeptide of the invention can be chosen by those skilled in theart, and the purification monitored, for example, by staining SDS-PAGEgels containing protein samples, by immunodetection methods such asWestern blotting and ELISA, and by functional assay of immunogenicactivity of flagellin or a TLR5 activity of TLR5.

An immunomodulatory flagellin peptide, polypeptide, TLR5 or fragmentsthereof can be modified, for example, to increase polypeptide stability,alter an activity, facilitate detection or purification, or render theenzyme better suited for a particular application, such as by alteringsubstrate specificity. Computer programs known in the art can be used todetermine which amino acid residues of a immunomodulatory flagellinpeptide, flagellin polypeptide or TLR5 can be modified as describedabove without abolishing a corresponding activity (see, for example,Eroshkin et al., Comput. Appl. Biosci. 9: 491-497 (1993)). In addition,structural and sequence information can be used to determine the aminoacid residues important for activity. For example, a comparisons offlagellin amino acid sequences, such as that shown in FIG. 7 can provideguidance in determining amino acid residues that can be altered withoutabolishing flagellin or flagellin peptide activity by indicating aminoacid residues that are conserved across species. Conserved regions offlagellin are well known in the art and have been described, forexample, in Mimori-Kiyosue, et al., J. Mol. Viol. 270: 222-237, (1997).A crystal structure of flagellin can also provide guidance for makingflagellin modifications (Samatey et al. Nature, 410: 331-337 (2001)).Similarly, amino acid sequence comparisons between the disclosed murineTLR5, TLR5s of other species, and other toll-like receptor familymembers can provide guidance for determining amino acid residuesimportant for activity.

An isolated TLR5 is a TLR5 removed from one or more components withwhich it is naturally associated. Therefore, an isolated TLR5 can be acell lysate, cell fraction, such as a membrane fraction, or a purifiedTLR5 polypeptide. An isolated TLR5 can include a liposome or othercompound or matrix that stabilizes or promotes an active conformation ofthe receptor.

For treating or reducing the severity of a pathological condition a TLR5agonist or antagonist, immunomodulatory flagellin peptide, polypeptideor modification thereof, including a vaccine, can be formulated andadministered in a manner and in an amount appropriate for the conditionto be treated; the weight, gender, age and health of the individual; thebiochemical nature, bioactivity, bioavailability and side effects of theparticular compound; and in a manner compatible with concurrenttreatment regimens. An appropriate amount and formulation for aparticular therapeutic application in humans can be extrapolated basedon the activity of the compound in recognized animal models of theparticular disorder.

Animal models of aberrantly proliferative diseases can be used to assessa formulation of compound, including a vaccine or adjuvant containing animmunomodulatory flagellin peptide, polypeptide or modification thereof,for an amount sufficient to induce an immune response or amelioratedisease symptoms. Animal models of such pathological conditions wellknown in the art which are reliable predictors of treatments in humanindividuals for include, for example, animal models for tumor growth andmetastasis, infectious diseases and autoimmune disease.

There are numerous animal tumor models predictive of therapeutictreatment which are well known in the art. These models generallyinclude the inoculation or implantation of a laboratory animal withheterologous tumor cells followed by simultaneous or subsequentadministration of a therapeutic treatment. The efficacy of the treatmentis determined by measuring the extent of tumor growth or metastasis.Measurement of clinical or physiological indicators can alternatively oradditionally be assessed as an indicator of treatment efficacy.Exemplary animal tumor models can be found described in, for example,Brugge et al., Origins of Human Cancer, Cold Spring Harbor LaboratoryPress, Plain View, New York, (1991).

Similarly, animal models predictive for infectious disease also follow asimilar approach. Briefly, laboratory animals are inoculated with aninfectious agent and the progression of the infection is monitored by,for example, clinical symptoms, growth culture of the agent from aninfected tissue sample or biopsy in the presence or absence of thetherapeutic treatment. The reduction in severity of the diagnosticindicator is indicative of the efficacy of the treatment. A variety ofanimal models for infectious diseases are well known to those skilled inthe art.

One animal model predictive for autoimmune diseases is Experimentalallergic encephalomyelitis (EAE), also called experimental autoimmuneencephalomyelitis. Although originally characterized as a model forneurological autoimmune disease such as human multiple sclerosis, theuse of this model to predict treatments of other autoimmune diseases hasbeen widely accepted. EAE is induced in susceptible animals by activeimmunization with myelin basic protein (MPB) or by passive transfer ofMBP-specific T helper lymphocytes. Progression of the disease ischaracterized by chronic relapsing paralysis and central nervous systemdemyelination, which can be monitored by observation or by immunologicaldeterminants such as delayed-type hypersensitivity (DTH; a measure ofcell mediated immunity) response to the immunogen. Efficacy of atherapeutic treatment is compared to progression of the disease in theabsence of treatment. A reduction in severity of EAE symptoms orimmunological determinants in treated animals is indicative of theefficacy of the therapeutic treatment. For a review of autoimmunedisease models see, for example, Urban et al., Cell, 54: 577-592 (1988);Brostoff et al., Immunol. Ser. 59: 203-218 (1993) and U.S. Pat. Nos.5,614,192 and 5,612,035.

A growing number of human diseases have been classified as autoimmuneand include, for example, rheumatoid arthritis, myasthenia gravis,multiple sclerosis, psoriasis, systemic lupus erythmatosis, autoimmunethyroiditis, Graves' disease, inflammatory bowel disease, autoimmuneuveoretinitis, polymyositis and diabetes. Animal models for many ofthese have been developed and can be employed analogously as the EAEmodel described above predictive assessment of therapeutic treatmentsusing the compounds, vaccines and adjuvants in the methods of theinvention. Other reliable and predictive animal models are well known inthe art and similarly can be used to assess a compound formulation,including vaccine and adjuvant formulations containing animmunomodulatory flagellin peptide, polypeptide or modification thereof.

The total amount of a compound including an immunomodulatory flagellinpeptide, polypeptide or modification thereof, that modulates aTLR5-mediated immune response can be administered as a single dose or byinfusion over a relatively short period of time, or can be administeredin multiple doses administered over a more prolonged period of time.Additionally, a compound can be administered in a slow-release matrix,which can be implanted for systemic delivery at or near the site of thetarget tissue.

A compound that modulates a TLR5-mediated immune response can beadministered to an individual using a variety of methods known in theart including, for example, intravenously, intramuscularly,subcutaneously, intraorbitally, intracapsularly, intraperitoneally,intracistemally, intra-articularly, intracerebrally, orally,intravaginally, rectally, topically, intranasally, or transdermally.

A compound that modulates a TLR5-mediated immune response can beadministered to a subject as a pharmaceutical composition comprising thecompound and a pharmaceutically acceptable carrier. The choice ofpharmaceutically acceptable carrier depends on the route ofadministration of the compound and on its particular physical andchemical characteristics. Pharmaceutically acceptable carriers are wellknown in the art and include sterile aqueous solvents such asphysiologically buffered saline, and other solvents or vehicles such asglycols, glycerol, oils such as olive oil and injectable organic esters.A pharmaceutically acceptable carrier can further containphysiologically acceptable compounds that stabilize the compound,increase its solubility, or increase its absorption. Suchphysiologically acceptable compounds include carbohydrates such asglucose, sucrose or dextrans; antioxidants, such as ascorbic acid orglutathione; chelating agents; and low molecular weight proteins. Asdescribed above in reference to vaccines, such routes of administrationare also applicable to administration of an immunomodulatory flagellinpeptide, polypeptide or modification thereof.

In addition, a formulation of a compound that modulates a TLR5-mediatedimmune response can be incorporated into biodegradable polymers allowingfor sustained release of the compound, the polymers being implanted inthe vicinity of where drug delivery is desired, for example, at the siteof a tumor or implanted so that the compound is released systemicallyover time. Osmotic minipumps also can be used to provide controlleddelivery of specific concentrations of a compound through cannulae tothe site of interest, such as directly into a tumor growth or other siteof a pathology involving a perturbation state. The biodegradablepolymers and their use are described, for example, in detail in Brem etal., J. Neurosurg. 74: 441-446 (1991). These methods, in addition tothose described above in reference to vaccines, are applicable toadministering an immunomodulatory flagellin peptide, polypeptide ormodification thereof to induce an immune response.

The methods of treating a pathological condition additionally can bepracticed in conjunction with other therapies. For example, for treatingcancer, the methods of the invention can be practiced prior to, during,or subsequent to conventional cancer treatments such as surgery,chemotherapy, including administration of cytokines and growth factors,radiation or other methods known in the art. Similarly, for treatingpathological conditions which include infectious disease, the methods ofthe invention can be practiced prior to, during, or subsequent toconventional treatments, such as antibiotic administration, againstinfectious agents or other methods known in the art. Treatment ofpathological conditions of autoimmune disorders also can be accomplishedby combining the methods of the invention for inducing an immuneresponse with conventional treatments for the particular autoimmunediseases. Conventional treatments include, for example, chemotherapy,steroid therapy, insulin and other growth factor and cytokine therapy,passive immunity and inhibitors of T cell receptor binding. The methodsof the invention can be administered in conjunction with these or othermethods known in the art and at various times prior, during orsubsequent to initiation of conventional treatments. For a descriptionof treatments for pathological conditions characterized by aberrant cellgrowth see, for example, The Merck Manual, Sixteenth Ed, (Berkow, R.,Editor) Rahway, N. J., 1992.

As described above, administration of a compound, immunomodulatoryflagellin peptide, flagellin polypeptide or modification thereof can be,for example, simultaneous with or delivered in alternativeadministrations with the conventional therapy, including multipleadministrations. Simultaneous administration can be, for example,together in the same formulation or in different formulations deliveredat about the same time or immediately in sequence. Alternatingadministrations can be, for example, delivering an immunomodulatoryflagellin peptide or polypeptide formulation and a conventionaltherapeutic treatment in temporally separate administrations. Asdescribed previously, the temporally separate administrations of acompound, immunomodulatory flagellin peptide, polypeptide ormodification thereof, and conventional therapy can similarly usedifferent modes of delivery and routes.

The invention provides a method of using a signal produced in responseto flagellin binding to TLR5 to detect bacterial contamination in asample. The method can be used to detect picogram amounts of flagellinin a sample.

Food-born diseases resulting from the presence of harmful bacteriaaccount for 325,000 hospitalizations and 5,000 deaths each year in theUnited States (National Institutes of Health, Foodborne Diseases NIAIDFact Sheet). The U.S. Centers for Disease Control and Prevention (CDC)estimates that 1.4 million people in the United States are infected eachyear with Salmonella. Other bacterial pathogens that cause pathologicalconditions characterized by symptoms ranging from intestinal discomfortto severe dehydration, bloody diarrhea and even death, includeenterohemorrhagic E. coli, such as strains designated O157: H7 andO26:H11, Campylobacter strains such as C. jejuni, and Shigella strainssuch as S. flexneri.

All of these bacterial strains are flagellated, and therefore expressflagellin polypeptides. For example, the amino acid sequences offlagellins from Salmonella, E. coli, Campylobacter, Shigella strains areshown in FIG. 7. The methods of the invention for detecting flagellinpolypeptides contained in samples suspected of bacterial contaminationcan be applied to quality assurance protocols for preparation of foodsand numerous other applications.

The invention also provides a bioassay for detecting bacterialcontamination in a sample. The method involves, (a) contacting thesample with a TLR5 under conditions wherein binding of a flagellinpolypeptide or fragment thereof in the sample to the TLR5 produces apredetermined signal, (b) determining the production of thepredetermined signal in the presence and absence of the sample, and (c)comparing the predetermined signal in the presence of the sample with apredetermined signal in the absence of the sample, wherein a differencebetween the predetermined signals in the presence and absence of thesample indicates that the sample contains flagellin.

The methods of the invention for detecting bacterial contamination arebased on the finding disclosed herein that flagellin is a ligand forTLR5. Therefore, a flagellin molecule in a sample can bind to a TLR5 andelicit the production of a predetermined signal. A predetermined signalproduced by TLR5 in a particular assay system is compared in thepresence and absence of a sample known or suspected of containing abacterial contaminant. A sample known to be free of flagellin can beused as a negative control, while a sample containing a knownconcentration of flagellin, flagella or bacteria having flagella can beused as a positive control.

A sample to be tested for the presence of flagellin can be any materialthat is suspected of being contaminated with a gram-positive orgram-negative flagellated bacterium. For example, the method fordetermining the presence of flagellin can be performed using a sample ofa biological fluid, cell, tissue, organ or portion thereof, such as asample of a tissue to be used for preparing a product, a product forhuman or animal consumption, such as a food or pharmaceuticalpreparation, and a product for external application or administration byany route to an animal.

A variety of predetermined signals produced by a TLR5, as discussedabove and in the Examples herein, can be used to detect the binding andactivation of a TLR5 by a flagellin molecule present in a sample. Avariety of methods known in the art, including those described hereincan be used to detect a predetermined signal produced by a TLR5.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Constitutively Active TLR5 Activates NF-kB and TNFa Production

This example shows activation of NF-kB and TNFa production in CHO cellsin response to constitutively active TLR5.

To determine if TLR5 activates NF-kB and TNFa production, the activityof a constitutively active form of TLR5 was examined in CHO cells.Constitutively active forms of TLR4 and TLR5 were generated by fusingthe extracellular domain of CD4 to the transmembrane and TIR domain ofTLR4 or TLR5 (Medzihitov, R. et al. Nature 388, 394-7 (1997); Ozinsky,A. et al., Proc. Natl. Acad. Sci. 97, 13766-13881 (2000)). CD4-TLR5 wasconstructed by fusing the murine CD4 extracellular domain (amino acids1-391) to the putative transmembrane and cytoplasmic domains of humanTLR5 (amino acids 639-859) and cloning into pEF6-TOPO (pEF6-mCD4-hTLR5).These chimeras, referred to as CD4-TLR4 and CD4-TLR5 were expressed inCHO cells.

For determining NF-kB activity in response to TLR5, CHO cells weretransiently transfected with expression vectors for CD4-TLR4, CD4-TLR5,or empty expression vector (control) together with an NF-kB luciferasereporter. NF-kB-induced luciferase activity was measured. CHO cells(CHO-K1) were obtained from ATCC (no. CRL.-9618) and grown in Ham's F-12medium supplemented with 10% FBS, L-glutamine, penicillin, andstreptomycin. CHO cells were transfected by electroporation as describedpreviously (Underhill, D. M. et al., Nature, 401, 811-5 (1999)), with 1mg of the indicated TLR expression vector, 1 mg of ELAM-fireflyluciferase, 0.1 mg of TK-renilla luciferase (Promega). Cells were platedon 96-well plates at 100,000 cells/well, and incubated overnight at 37°C., 5% CO₂. Firefly and renilla luciferase activities were measuredusing the Dual Luciferase Assay System (Promega, Madison, Wis.).Luciferase activity is expressed as a ratio of NF-kB-dependentELAM-firefly luciferase activity divided by control thymidinekinase-renilla luciferase activity (relative luciferase units).

For determining TNFα production in response to TLR5, RAW-TTIO Macrophagecells were transfected with a CD4-TLR5 expression vector, and theproduction of TNFa was measured by flow cytometry, as describedpreviously (Ozinsky, A. et al. Proc. Natl. Acad. Sci. 97, 13766-13771(2000)). Transfections were performed by electroporation using 10 mg ofpEF6-mCD4-hTLR5, and 18 hours later the cells were incubated with 5mg/ml of brefeldin A for 4 hours to accumulate intracellular pools ofnewly synthesized TNFa. Cells were fixed, permeabilized, stained for theexpression of CD4 (anti-CD4-FITC, Pharmingen) and TNFa (anti-murineTNFa-PE, Pharmingen), and analyzed on a FACscan (Beckton-Dickenson).FACS data were analyzed with WinMDI (Joseph Trotter, Scripps ResearchInstitute, La Jolla, Calif.). Cells were gated to exclude dead cells andfor expression of CD4.

FIG. 1 shows that expression of CD4-TLR5 induced NF-kBactivation-mediated luciferase production in CHO cells (FIG. 1 a) andTNFa production in mouse macrophages (FIG. 1 b). In FIG. 1 b, the dottedline indicates TNFa produced in cells not expressing CD4-TLR5, and thesolid line indicates TNFa produced in cells expressing CD4-TLR5. Thus,homo-oligomerization of the TLR5 signaling domain induces a cellularsignal characterized by the induction of NF-KB activity and productionof TNFa.

EXAMPLE II Bacterial Culture Supernatants Contain TLR5-StimulatingActivity

This Example shows that bacterial culture supernatants containTLR5-stimulating activity.

CHO cells expressing human TLR5 and a luciferase-linked reporter wereused to screen for PAMPs recognized by the receptor. PAMPS testedincluded LPS, lipopeptide, yeast, and extracts from E. coli,Pseudomonas, and Listeria. CHO cells were transiently transfected withTLR2, TLR5, or empty expression vectors together with a NF-kB luciferasereporter. The cells were treated with 100 ng/ml LPS, 100 ng/mllipopeptide, 10⁷ yeast particles/ml, or untreated (control), andluciferase activity was measured. The cells were treated with 67 mg/mlof supernatant from the indicated saturated bacterial cultures, or LBalone (control), and the luciferase activity was measured. Data arerepresentative of 3 independent experiments.

Human TLR5 and TLR2 were generated by PCR from cDNA derived from humanperipheral blood mononuclear cells and cloned into pEF6-TOPO(Invitrogen) (pEF6-hTLR5 and pEF6-hTLR2). Murine TLR5 was generated byPCR using cDNA derived from RAW-TT1O cells and cloned into pEF6(pEF6−mTLR5).

For luciferase assays, CHO cells were transfected by electroporation asdescribed above, with 1 mg of the indicated TLR expression vector, 1 mgof ELAM-firefly luciferase, 0.1 mg of TK-renilla luciferase (Promega,Madison, Wis.). The medium was replaced with medium containing thestimuli at the indicated concentration/dilution. Bacterial lipopeptideand PAM₃CSK₄, were obtained from Roche, LPS (Salmonella minnesota R595)was from List, and yeast particles (zymosan) were from Molecular Probes(Eugene, Oreg.). Cells were stimulated for 5 hours at 37° C., andfirefly and renilla luciferase activities were measured using the DualLuciferase Assay System (Promega).

For preparation of bacterial supernatants, bacteria were grown either inLuria broth (LB) (Escherichia coli TOP 10 (Invitrogen), Salmonellaminnesota (ATCC#49284), mutant Salmonella typhimurium (TH4778fliB−fliC+), TH2795 (fliB− fliC−), (Dr. Kelly Hughes, University ofWashington), or grown in trypticase soy broth (TSB) (Listeriamonocytogenes (10403, gift of Dr. Daniel Portnoy, UCSF), Listeriainnocua (ATCC#33090), Bacillus subtilis, and Pseudomonas aeruginosa(Susan R. Swanzy, University of Washington)). Bacteria were grown tosaturation (about 16 hours, 37° C. with vigorous aeration). Thebacterial culture supernatants were centrifuged for 30 minutes at2000×g, filtered (0.2 mM), and stored at 4° C. prior to use. For flaAtransfections, E. coli TOP10 containing pTrcHis2-flaA orpTrcHis2-flaArev were selected from bacterial plates and grown to OD₆₀₀of 0.6 in LB with 100 ug/ml ampicillin and 1% w/v glucose. The bacteriawere centrifuged for 30 minutes at 2000×g, and split into two LBcultures, one containing 100 mg/ml ampicillin and 1% w/v glucose (torepress flaA) and the other containing 100 mg/ml ampicillin and 1 mMIPTG (to induce flaA). Samples were taken at 4 hours after induction,centrifuged 5 min at 10,000×g, and the supernatants stored at 4° C.before use.

TLR5 did not respond to any of the PAMPs known to stimulate TLRpathways, such as LPS, lipopeptide, yeast cell wall, or peptidoglycan,while CHO cells transfected with TLR2 were stimulated by lipopeptide,yeast cell wall, and peptidoglycan (FIG. 2 a). However, TLR5-stimulatingactivity was detected in culture supernatants of a variety ofGram-positive and Gram-negative bacteria (FIG. 2 b). TheTLR5-stimulating activity of Gram-positive bacteria was not enhanced byco-expression of CD 14. Interestingly, the TOP10 strain of E. coli hadvery little TLR5 activity (FIG. 2 b), and was used in subsequentreconstitution experiments (see below). Experiments using murine TLR5yielded similar results.

Thus, the activity of TLR5 was stimulated by a component of bacterialculture supernatants, but not by PAMPs known to stimulate other tolllike receptor family members.

EXAMPLE III Purification of TLR5-Stimulating Activity from L.monocytogenes Culture Supernatant

This Example shows the purification of TLR5-simulating activity from L.monocytogenes culture supernatant.

The biological activity recognized by TLR5 was determined to be TCAprecipitable, phenol soluble, and sensitive to proteinase K and trypsindigestion. To identify the bacterial components that stimulate TLR5, thesupernatant from a saturated L. monocytogenes culture was concentrated,fractionated by reverse-phase chromatography, and each fraction wasassessed for TLR5-stimulating activity in CHO cells (FIG. 3 a).

For assessing the TLR-stimulating activity of FPLC fractions, CHO cellswere transfected as described in Example I with the addition of 0.1 mgof pNeo/Tak (Underhill et al., Nature 401, 811-5 (1999)), and stablepopulations of cells expressing the indicated TLR with the luciferasereporters were selected in 100 mg/ml G418. These cells were plated on96-well plates at 100,000 cells/well and incubated overnight.

For the purification of the TLR5-stimulating activity, saturated L.monocytogenes culture (200 ml of TSB) was centrifuged, and thesupernatant was enriched for molecules larger than 30 kDa byultrafiltration (Ultrafree-15 filter unit with Biomax-30 membrane,Millipore). The buffer was changed to 100 mM Tris pH 7.5, and the volumewas adjusted to 5 ml. The sample was loaded onto a HR5/10 reverse-phasechromatography column (AP Biotech) and run at 0.3 ml/min. Reverse-phasechromatography was performed with the indicated elution profile usingthe following buffers: (A) initial buffer, 0.1% TFA in water, (B) finalbuffer, 0.1% TFA in acetonitrile. Fractions were collected at 3-minuteintervals. FPLC fractions (50 ml) were separated on a 10% SDS-PAGE gel.

As shown in FIG. 3 a, CHO cells expressing an NF-KB luciferase reporterand TLR5 were stimulated with reverse-phase FPLC fractions, andTLR5-mediated NF-kB luciferase activity was measured. The fractionnumbers correspond to 3 minute fractions of reverse-phase FPLC elutedwith a non-linear gradient of buffer B as shown. Fraction number “N” iscontrol LB growth medium and “P” is the L. monocytogenes culturesupernatant prior to chromatography. Fractions containing backgroundactivity (1), low activity (2) and high activity (3) as indicated inFIG. 3 a were analyzed by SDS-PAGE and silver stain. Silver staining wasperformed according to established methods. Two bands with apparentmolecular masses of 30-34 kDa were clearly enriched in the fractioncontaining the highest level of TLR5-stimulating activity (FIG. 3 b,Lane 3). Proteins eluted-from regions A, B, and C of the SDS-PAGE gel,as indicated in FIG. 3 b were assayed for TLR5-mediated NF-kB activationin CHO cells. In FIG. 3 c, “Listeria” indicates L. monocytogenes culturesupernatant. One of these bands, (FIG. 3 b, band A), wastrypsin-treated, subject to microcapillary HPLC-tandem massspectrometry, and identified by comparison of peptide tandem massspectra to sequences in a non redundant protein database using thecomputer program, SEQUEST27 (FIG. 4 a). TLR5-stimulating activity wasnot recovered from any other section of the gel. Thus, aTLR5-stimulating activity was purified from culture supernatants from L.monocytogenes.

EXAMPLE IV Flagellin is a TLR5 Stimulus

This example shows that flagellin is a TLR5 stimulus purified fromculture supernatants from L. monocytogenes.

As described above, a TLR5-stimulating activity was purified from L.monocytogenes culture supernatants using HPLC. The isolated polypeptideof band A in FIG. 3 b was trypsinized and identified by microcapillaryHPLC-tandem mass spectrometry. Peaks corresponding to L. monocytogenesflagellin peptides are indicated in FIG. 4 a. Five sequences wereidentified (FIG. 4 a) that correspond to flagellin, the product of theflaA gene of L. monocytogenes (Genbank Q02551). The location of thesesequences within the protein is indicated in FIG. 4 b. Band B of FIG. 3b also is flagellin, which migrates as a doublet of approximately 30 kDaon SDS-PAGE (FIG. 3 b).

For analysis, bands A and B were excised from SDS-PAGE gels, dehydratedwith acetonitrile, dried under reduced vacuum, and trypsin (12.5 ng/mL)was infused into the gel. The gel slice was allowed to incubate on icefor 45 min in the presence of trypsin and then excess trypsin removedand replaced with 50 mM ammonium bicarbonate and the gel slice incubatedovernight at 37° C. Peptides were extracted by 3 washes with 5% aceticacid in 50% aqueous acetonitrile. The extractions were pooled andconcentrated by vacuum centrifugation. The peptides were injected onto aC18 peptide trap cartridge (Michrom BioResources, Inc. Auburn, Calif.),desalted, and then injected onto a 75 mm (internal diameter)×10 cmmicro-capillary HPLC column (Magic C18; 5-mm packing; 100 A pore size;Michrom BioResources, Inc. Auburn, Calif.). The sample injection wasmade using a FAMOS autosampler (LCPackings, San Francisco, Calif.)coupled with an Agilent HP 1100 Pump. Peptides were separated by alinear gradient of acetonitrile, and subjected to collision induceddissociation using an electrospray ionization-ion trap mass spectrometer(ESI-ITMS; ThermoQuest, San Jose, Calif.) in data-dependent mode withdynamic exclusion (Goodlett, et al. Anal. Chem. 72, 1112-1118 (2000)).Protein identification was accomplished by use of the SEQUEST computerprogram (Eng et al., J. Am. Soc. Mass. Spectrom., 5: 976-989 (1994)).

CHO cells expressing an NF-kB luciferase reporter and TLR5 or TLR2 werestimulated with 100 ml/ml Listeria supernatant or 33 mg/ml purifiedSalmonella flagellin. Flagellin was purified from Salmonella typhimurium(TH4778fliB− fliC+) by the procedure of Ibrahim et al., J. Clin.Microbiol., 22: 1040-1044 (1985). As shown in FIG. 4 c, flagellinstimulated TLR5-expressing CHO cells, but not TLR2-expressing CHO cells.The mean and standard deviation of quadruplicate samples are indicated.CHO cells were transfected as described in above Examples with theaddition of 0.1 mg of pNeo/Tak, and stable populations of cellsexpressing the indicated TLR with the luciferase reporters were selectedin 100 mg/ml G418. These cells were plated on 96-well plates at 100,000cells/well, incubated overnight, and processed in luciferase assays asdescribed above.

The observation that flagellin is the TLR5 ligand also is supported bythe finding that the flagellated bacteria, L. monocytogenes and P.aeruginosa, stimulate TLR5, while the TOP10 strain of E. coli, that haslost its flagella, does not (FIG. 2 b). Similarly, TLR5-stimulatingactivity was found in B. subtilis, L. innocua, S. typhimurium and S.minnesota, all flagellated bacteria, while non-flagellated bacteria suchas H. influenza, did not activate TLR5. Thus, the TLR5-stimulatingactivity purified from L. monocytogenes culture supernatants wasidentified as flagellin by tandem mass spectrometry.

EXAMPLE V Flagellin Expression in Bacteria ReconstitutesTLR5-Stimulating Activity

This Example shows that flagellin expression in bacteria reconstitutesTLR-stimulating activity, and deletion of flagellin genes abrogatesTLR5-stimulating activity.

To confirm that flagellin is the sole TLR5 ligand in bacteria, E. coli(TOP1O) that secrete little TLR5 activity (FIG. 2 b) were transformedwith the cDNA of L. monocytogenes flagellin (flaA) under the control ofan inducible promoter. TLR-expressing CHO cells were stimulated for 5hours with E. coli culture supernatants (67 ml/ml) in which expressionof L. monocytogenes flagellin was induced or repressed. In the controlsample, CHO cells were stimulated with supernatants from induced E. colicontaining the L. monocytogenes flagellin gene cloned in the reverseorientation. Supernatants of E. coli that were induced to express L.monocytogenes flaA contained substantial TLR5-stimulating activity (FIG.5 a), whereas supernatants from E. coli in which expression wasrepressed, or from E. coli expressing flaA in the reverse orientation,contained little TLR5 activity in CHO cells expressing an NF-kBluciferase reporter and TLR5 (FIG. 5 a) or TLR2 (FIG. 5 b). CHO cellsexpressing an NF-kB luciferase reporter and TLR5 (c) or TLR2 (d) werestimulated for 5 hours with culture supernatants (100 ml/ml) from S.typhimurium lacking one copy of flagellin (FliB− fliC+) or both copiesof flagellin (FliB+ FliC+). Control is stimulation with LB medium. Themean and standard deviation of quadruplicate samples are indicated.

CHO cells were transfected with TLR2 and TLR5 expression plasmids asdescribed above with the addition of 0.1 mg of pNeo/Tak, and stablepopulations of cells expressing the indicated TLR with the luciferasereporters were selected in 100 mg/ml G418. These cells were plated on96-well plates at 100,000 cells/well, incubated overnight, and processedin luciferase assays as described above.

L. monocytogenes flagellin is not recognized by TLR2, since supernatantsfrom E. coli expressing flaA did not show enhanced TLR2-dependentstimulation of CHO cells relative to supernatants from E. coli withrepressed flaA expression (FIG. 5 b). In addition to the experimentsthat demonstrate reconstitution of TLR5-stimulating activity by theexpression of flagellin, a bacterium from which flagellin had beendeleted was tested. It was observed that TLR5-stimulating activity wasabrogated in the flagellin deleted strain. S. typhimurium possess twogenes for flagellin, fliB and fliC (Fujita, J., J. Gen Microbiol. 76:127-34 (1973)). Culture supernatants of fliB− fliC+S. typhimuriumcontained TLR5-stimulating activity, while culture supernatants from S.typhimurium lacking both flagellins (fliB− fliC−) expressed noTLR5-stimulating activity (FIG. 5 c). The lack of both flagellin geneshad no effect on TLR2-stimulating activity (FIG. 5 d). The observedTLR2-stimulating activity found in S. typhimurium supernatants mostlikely was due to bacterial lipoproteins (Underhill, et al., Nature,401: 811-5 (1999); Brightbill et al., Science, 285: 732-6 (1999)). Theseresults indicate that flagellin is the sole TLR5-stimulating activitypresent in S. typhimurium culture supernatant. Thus, TLR5-stimulatingactivity was elicited by introducing the flagellin gene into anon-flagellated bacterium, and abrogated by deleting the flagellin genesfrom a flagellated bacterium.

EXAMPLE VI Flagellin-Induced System IL-6 Production in Mice

This example shows that TLR signaling is required for the in vivo immuneresponse to flagellin.

To determine if TLR signaling is required for the in vivo immuneresponse to flagellin, wild type mice and mice lacking a component ofthe TLR5 signal transduction pathway, MyD88, were injected withflagellin and systemic IL-6 production was monitored. MyD88 is anadaptor protein required for TLR5-mediated signal transduction (AderemA., et al., Nature, 406: 782-787, (2000); Brightbill, H. D. and Modlin.R. L., Immunology, 101: 1-10, (2000)).

MyD88^(-/-) mice (129/SvJ×C57B1/6 background) were backcrossed for threegenerations with C57B1/6 mice (Adachi, O. et al., Immunity, 9: 143-150(1998)). Mice from the F₃ generation (MyD88^(-/-), n=5) and littermatecontrols (MyD88+/+, n=5) were injected i.p. with 30 mg purifiedflagellin in 0.5 cc of saline. Blood was sampled at 0, 1, 2, 4 and 8hours after injection, and IL-6 levels were determined by ELISA (Duoset,R&D Systems, Minneapolis, Minn.).

FIG. 6 shows that flagellin induced systemic IL-6 within 2 h in wiletype mice. By contrast, mice deficient in MyD88 were completelyunresponsive to flagellin. Therefore, flagellin stimulates TLR5-mediatedresponses in vivo.

EXAMPLE VII TLR-5 Recognition of a Conserved Site on Flagellin

This example shows that that TLR5 recognizes a conserved site onflagellin required for protofilament formation and bacterial motility.

TLR5 Recognizes Evolutionarily Conserved Domain of Flagellin. Flagellinis known to undergo N-methylation of lysine residues, and glycosylationin some bacteria. Using Salmonella typhimurium flagellin expressed inand secreted from Chinese hamster ovary (CHO) cells, we determined thatbacteria-specific post-translational modifications of flagellin were notrequired for TLR5 recognition.

To define the region of the flagellin monomer recognized by TLR5, wemade flagellin deletion mutants and tested whether they were able toactivate TLR5. Briefly, the D1-99 mutant was made by PCR amplificationof S. typhimurium fliC gene (GenBank accession no. D13689) encodingamino acids 100-494, and adding an ATG codon to the 5′ end of the100-494 coding region. This PCR fragment was cloned into pTrcHIS2(Invitrogen), and transformed into flagellin-negative (Hayashi, F. etal., Nature, 410: 1099-1103 (2001)) TOP10 E. coli cells (Invitrogen).Construction of S. typhimurium BC379 strain, and the ptrcfliC::i31plasmids were performed by placing fliC::i31 alleles in-frame insertionof an additional 31 codons (non-fliC DNA) into the fliC gene (Manoil, C.et al., J. Mol. Biol., 267: 250-263 (1997)). The unique BamHI sitelocated within the 31-codon sequence allowed restriction and religationof different ptrcfliC::i31 plasmids to create new plasmids encodingFliC::i31 with internal deletions between insertion sites. Bacterialcultures, grown overnight at 37° C. with 50 ug/ml ampicillin and 1 mMIPTG, were probe-sonicated for 60 sec to release and disperse flagellinfrom bacterial cells.

CHO K1 cells (ATCC) were grown in HAM's F-12 medium with penicillin,streptomycin, L-glutamine and 10% fetal calf serum (HyClone). RAW 264.7cells were grown in RPMI 1640 with penicillin, streptomycin, L-glutamineand 10% fetal calf serum. NF-kB luciferase reporter assays werepreformed as follows: CHO K1 cells were transfected with either thehuman or mouse TLR5 cDNA cloned into the pEF6 V5/His TOPO vector(Invitrogen), ELAM-LUC (Underhill, et al. Proc. Natl. Acad. Sci. USA,96: 14459-14463 (1999)) and pRL-TK (Promega) plasmids, selected withblasticidin, and cloned by limiting dilution. Stable clones werestimulated for 4-5 h, and assayed for luciferase activity. All assayswere done in triplicate, and each experiment was repeated at least threetimes. Fold-induction was calculated by dividing the luciferase valuesfor the test conditions by the relative luciferase value for the controlcondition.

FIG. 8 shows the results of these deletion mutants. In FIG. 8(a) CHOcells expressing human TLR5 and an NF-κB luciferase reporter werestimulated with bacterial sonicates (approximately 10⁸ cells/ml) fromcells expressing no flagellin (BC379), WT flagellin, or an N-terminal100 amino acid flagellin deletion mutant (Δ1-99). Shown is the foldinduction of luciferase activity relative control cells (BC379). FIG.8(b) is an immunoblot of bacterial sonicates from FliC deletion mutants,showing the detection of FliC with rabbit anti-FliC antiserum. FIG. 8(c)shows FliC deletion mutants were tested as in (a) for theirTLR5-stimulatory capacity; data shown is representative of at leastthree independent experiments. FIG. 8(d) illustrates the position of thedeletion mutants tested is shown on the ribbon diagram of the flagellinstructure (Samatey, F. A. et al., Nature, 410: 331-337 (2001)). In theribbon diagram of flagellin, the D1 domain consists of the red and bluecolored segments, the D2 domain is colored green, and the D3 domain iscolored yellow. The overlying green areas designate the deletions thathad no effect on TLR5 recognition, and the red areas designate deletionsthat abrogated TLR5 recognition.

Deletion of the amino-terminal 99 amino acids of the S. typhimurium FliCflagellin monomer prevented TLR5 recognition (FIG. 8 a). There was nodetectable stimulation of vector control transfected CHO cells (data notshown). Deletions that removed amino acids 416-444 within the C-terminusof FliC were sufficient to abrogate TLR5 recognition (FIG. 8 b, c andd). These D1 domain polypeptide regions are highly conserved amongstbacteria, and are essential for motility. In contrast, deletion of FliCresidues 444-492, within the C-terminal D1 domain, or D3 domain residues185-306, within the hypervariable domain, had no effect. The D3 domainis exposed at the surface of the flagellar filament, is not required formotility, and is a common target for antibody responses. Its high degreeof variability suggests that the D3 domain has evolved to permitconsiderable structural heterogeneity in order to evade adaptive immuneresponses.

We refined the TLR5 recognition site using a panel of 23 S. typhimuriumfliC transposon insertion mutants. These mutants were generated using aTn lacZ/in transposon system (Manoil, C., et al., J. Mol. Biol., 267:250-263 (1997)), which results in the insertion of an in-frame 31 aminoacid polypeptide into the flagellin sequence.

The results of this analysis are shown in FIG. 9 and indicate that S.typhimuirium FliC with insertions in the conserved D1 domain afterresidues 93, 166, 168, 416 and 424, abrogated TLR5 recognition, whereasnone of the other 18 insertions had any effect (FIG. 9 c). Briefly, FIG.9(a) indicates the position of insertions tested on the ribbon diagramof the flagellin structure, with the red area highlighting the clusterof insertions that abrogate TLR5 recognition. FIG. 9(b) is an immunoblotfor flagellin demonstrating comparable amounts of S. typhimurium FliCflagellin for the control (WT) and insertion mutants. Insertion mutantsshown in FIG. 9(c) were tested as in FIG. 8, using CHO cells expressinghuman TLR5; data shown is representative of at least three independentexperiments. The amount of sonicated bacterial cells used to stimulateapproximately 10⁵ CHO cells in a 200 ul volume is indicated in thelegend. In combination, these studies mapped the TLR5 recognition siteon flagellin to a discrete region in the D1 domain (FIG. 9 a).

The above studies demonstrate that TLR5 recognizes a site on flagellincomprised of amino terminal residues 78-129 and 135-173, andcarboxyl-terminal residues 395-444. We compared the amino acid sequencesof flagellin molecules from bacteria with known TLR5-stimulatoryactivity (Ref. 13, 42 and inventor's observations). This narrowed downthe conserved regions of flagellin to amino acid residues 79-118, and408-439 (FIG. 10 a). By comparing the aligned sequences, we identifiedconserved amino acid residues within these regions that were likely tobe important for TLR5 recognition (FIG. 10 a). The non-alanine or-glycine residues in these regions were chosen as candidates forenergetically important contacts with TLR5 and 22 alanine mutations weremade in flagellin (FIG. 10 a). The proteins were purified, quantitated,and analyzed by SDS-PAGE and Coomassie blue stain to assess their purity(data not shown).

To generate the flagellin alanine mutants, the fliC gene was cloned intothe NcoI and HindIII sites of ptrc99a plasmid. Single amino acidmutations were made using a standard PCR mutagenesis strategy (Smith, K.D., et al., PCR Methods Appl., 2: 253-257 (1993)). All mutations wereverified by DNA sequencing. The mutant plasmids were transformed intothe BC696 (fliB−/fliC−) strain of S. typhimurium SL1344. BC696 wasconstructed by the method of Datsenko and Wanner (Datsenko, K. A., etal., Proc. Natl. Acad. Sci. USA, 97: 6640-6645 (2000)). Briefly, λred-mediated recombination was used to replace the fliC gene of S.typhimurium with a cassette encoding Kan^(R) flanked by FLP recognitiontarget (FRT), which was subsequently excised. The same procedure wasrepeated at the fliB allele to create BC696, which lacks both flagellingenes, which was confirmed by PCR, immunoblot and motility assays.Flagellin mutant protein expression in BC696 transformants was inducedby culture in the presence of 1 mM IPTG, and confirmed by immunoblot,using rabbit anti-FliC anti-serum (Difco) and a goat anti-rabbit horseradish peroxidase conjugate secondary (Jackson Immunolabs).

The results of this study are shown in FIG. 10 where panel (a) shows aClustalW alignment of flagellin protein sequences from TLR5-stimulatorybacteria. Residues that were mutated to alanine are indicated with anarrow (⇓). Dose-response curves demonstrate representative examples ofalanine point mutations that had no significant effect on TLR5recognition (FIG. 10 b), slightly reduced TLR5 recognition (FIG. 10 c)or substantially reduced TLR5 recognition (FIG. 10 d). FIG. 10(e) showsthe effective concentration for half-maximal TLR5 stimulation (EC₅₀)which was calculated for each point mutant and plotted as a bar graph.The mean EC₅₀±S.D. was determined from at least 3 independentexperiments. An asterisk (*) denotes the mutants with EC₅₀ that weresignificantly different from WT flagellin.

As shown in FIG. 10 b-d, the individual alanine mutants tested for theirability to stimulate TLR5 were found to segregate into three broadclasses: those that had no effect (FIG. 10 b), those that slightlyreduced TLR5 recognition (FIG. 10 c), or those that substantiallyreduced TLR5 recognition (FIG. 10 d). The effective flagellinconcentration required for 50% maximal stimulation (EC₅₀) was calculatedfor each point mutant, using results from at least three independentexperiments (FIG. 10 e). Of the 22 alanine mutants, nine did notsignificantly (p>0.05) affect TLR5 recognition, 3 (N100A, D412A, andR431A) significantly reduced TLR5 recognition by 50-75%.

(p<0.05), and 10 (L88A, Q89A, R90A, L94A, Q97A, E114, I411, L415, T420A,and L425A) significantly (p<0.001) reduced TLR5 recognition by 76-97%.No single mutation completely abrogated TLR5 recognition.

The alanine mutations also were found to affect bacterial motility.Bacterial motility assay were performed as follows. Bacteria werestab-inoculated into the center of motility plates (LB containing 0.3%Agar, with 50 ug/ml ampicillin and 1 mM IPTG). Cultures were incubatedupright at 37° C. for 12 h, and then photographed. The relative motilityof the bacteria harboring fliC alanine mutants was calculated bymeasuring the diameter of the bacterial swarm, and dividing this by thediameter for bacteria harboring the plasmid with WT fliC (% WTmotility). Motility was scored as scored as follows: ++, WT motility; +,30-80% WT motility; +/−, 5-30% WT motility; −, <5% WT motility.

Flagellin's amino- and carboxyl-terminal amino acids are evolutionarilyconserved, and many of these conserved residues are likely to beimportant for flagellar filament assembly and bacterial motility. Wetested the panel of flagellin alanine mutants for their effect onbacterial motility. Similar to TLR5 recognition, alanine mutants groupedinto three classes: three mutations did not alter motility, eightmutations reduced motility, and 11 mutations completely abrogatedbacterial motility. The results of this analysis are shown in Table 1.TABLE 1 Summary of the effect of alanine substitutions in flagellin onbacterial motility and TLR5 recognition. EC₅₀ (ng/ml) EC₅₀ Mutation¹Motility² TLR5³ Mean (S.D.) p-value WT ++ 3.4 (2.2) fliC-fljB- − E84A− + 5.5 (2.6) 0.086 L89A +/− − 48.0 (21)  <0.001 Q90A* ++ − 14.0 (5.9)<0.001 R91A* − − 25.0 (8.0) <0.001 R93A* + + 4.5 (2.0) 0.400 E94A* + +1.8 (0.9) 0.225 L95A* − − 29.0 (8.3) <0.001 V97A* + + 2.8 (1.4) 0.626Q98A* + − 19.0 (6.7) <0.001 N101A* ++ − 7.7 (7.8) 0.044 T103A* + + 4.9(2.3) 0.303 S105A* − + 4.7 (2.7) 0.394 D108A* − + 3.6 (1.1) 0.916 E115A− − 23.0 (4.4) <0.001 I412A − −− 115.0 (35)  <0.001 D413A − − 8.6 (2.6)<0.001 L416A ++ − 38.0 (13)  <0.001 T421A + − 25.0 (21)  <0.001 R422A− + 3.2 (1.2) 0.851 L426A + − 19.0 (10)  <0.001 Q430A − + 4.2 (1.6)0.521 R432A − − 12.0 (3.0) <0.001¹Residues located in convex intermolecular contact site [Samatey, 2001#519] are designated with and asterisk (*).²Bacterial motility is scored as follows: ++, WT motility; +, 30-80% WTmotility; +/−, 5-30% WT motility; −, <5% WT motility.³TLR5 Recognition is noted as follows: +, does not affect TLR5recognition; −, reduces TLR5 recognition 2-14 fold; −−, reduces TLR5recognition >30 fold.

The TLR5 recognition site, defined above, is buried within the core ofthe flagellar filament. This region is also predicted to be involved inaxial intermolecular contacts between individual flagellin monomers thatform the protofilament. We purified flagellin monomers and filamentsfrom S. typhimurium, and analyzed the protein preparations by SDS-PAGE(FIG. 11 a).

Briefly, purification of bacterial flagellin was performed growingovernight Salmonella typhimurium strain TH4778 (FliB−/FliC+; gift fromDr. Kelly Hughes, University of Washington) or BC696 harboring fliCexpression plasmids in LB medium (supplemented with 50 ug/ml ampicillinand 1 mM IPTG for transformed BC696 strains), and pelleted bycentrifugation. Cell pellets were washed once in PBS, resuspended inPBS, and sheared for 2 min at high speed in a Waring blender. Thesheared suspension was centrifuged for 10 min. at 8000×g, and thesupernatant was collected and centrifuged at 100,000×g for 1 h to pelletflagellin filaments. The pellets of flagellin filaments were resuspendedin PBS at 4° C. overnight, and centrifuged at 100,000×g for 1 h. Thiswash step was repeated twice.

Monomeric flagellin was prepared by resuspending the resulting pellet offlagellin filaments in PBS, heated to 70° C. for 15 min, and passedthrough a 100 kDa molecular weight cut-off filter (Amicon). Theresulting flagellin monomers were recovered from the filtrate, proteinconcentration was determined using the BCA assay (Pierce), and puritywas assessed by SDS-PAGE and Coomassie blue staining.

For purification of bacterial flagellin filaments, the preparation offlagellin filaments was extensively dialyzed against PBS using a 300 kDamolecular weight cut-off membrane (Pierce). The resulting flagellinfilaments were recovered, and protein concentration was determined usingthe BCA assay (Pierce), and purity was assessed by SDS-PAGE andCoomassie blue staining.

The electron microscopy studies described below were performed bymounting filamentous flagellin on a carbon-coated grid, negativelystained with tungsten phosphate with subsequent analysis by transmissionelectron microscopy.

Cross-linking studies were performed by dissolvingDithiobis[succinimidylpropionate] (DSP, Pierce) in DMSO to 25 mMimmediately prior to use. Filamentous flagellin (10 mg/ml in PBS) wascross-linked with 1 mM DSP for 30 min at room temperature; the reactionwas stopped by adding 50 mM glycine and incubating 15 min at roomtemperature. Mock-treated samples were incubated with an equal amount ofDMSO without DSP. The DSP treated protein was treated with 50 mM DTT for30 min at 37° C. to cleave the crosslinker. As a control, monomericflagellin was similarly reacted. The buffer solution for the abovereactions was changed back to PBS. Cross-linked and cleaved samples wereheated at 70° C. for 15 min to liberate any monomers for biologic assaysof TLR5-stimulatory activity.

FIG. 11 shows that TLR5 recognizes monomeric flagellin. FIG. 11(a) is aCoomassie-stained SDS-PAGE gels of monomeric and filamentous flagellinpreparations, showing equivalent amounts of the 50 kDa flagellin proteinin both preparations. FIG. 11(b) is an electron micrograph of flagellinfilaments. FIG. 11(c) shows the results of CHO cells expressing TLR5that were stimulated with either monomeric or filamentous flagellin.Fold-induction of NF-κB luciferase reporter was calculated for cellsstimulated with either the flagellin monomers or filaments to controlstimulated cells. FIG. 11(d) is a Coomassie-stained sDs-PAGE gels ofmonomeric and filamentous flagellin, untreated or treated withcross-linking agent DSP and/or reducing agent DTT, as described inMethods and indicated in the figure. The monomeric flagellin migrates asa 50 kDa protein (lanes 1 and 3). DSP modification of the flagellinmonomer results in a slight retardation in gel migration (lanes 2 and4). The unmodified flagellin filament is dispersed into 50 kDa monomers(lanes 5 and 7) when heated and denatured for SDS-PAGE. DSP cross-linkedflagellin filaments form large molecular complexes that cannot enter thegel (lane 6). Reduction of the DSP-crosslinked flagellin filaments withDTT liberates predominantly monomers, with a few higher molecular weightmultimers (lane 8). FIG. 11(e) shows the results of CHO cells expressingTLR5 that were stimulated with flagellin filaments that were leftuntreated, heated for 15 min at 70° C. to depolymerized into monomers,cross-linked with DSP and heated for 15 min at 70° C., or cross-linkedwith DSP, reduced with DTT, and heated for 15 min at 70° C.Fold-induction of NF-κB luciferase reporter was calculated for theflagellin preparation stimulated cells relative to control stimulatedcells. FIG. 11(f) shows the results of CHO cells expressing TLR5 thatwere stimulated with flagellin monomers that were left untreated,cross-linked with DSP, or cross-linked with DSP and reduced with DTT.Fold-induction of NF-KB luciferase reporter was calculated for cellsstimulated with the flagellin preparations relative to controlstimulated cells.

The results of the above analysis demonstrate that flagellin filamentsmigrated as monomers on SDS-PAGE gels, because they were depolymerizedby the heating step during preparation for electrophoresis (FIG. 11 a).The filamentous structure was confirmed by electron microscopy (FIG. 11b). Filamentous flagellin TLR5-stimulatory activity was reduced by 96%compared to monomeric flagellin (FIG. 11 c). Because some monomers werelikely to be liberated from the filament during preparation andstimulation, we stabilized the purified filaments by chemicalcross-linking with Dithiobis[succinimidylpropionate], DSP. Thecross-linked filaments could not be depolymerized, and were unable toenter the gel (FIG. 11 d, lane 6). When the polymerized form offlagellin was stabilized in this manner, TLR5 stimulatory activity wasreduced by greater than 99.5% (FIG. 11 e). DSP is a homobifunctional,thiol-cleavable, cross-linking agent. Reduction of this bond withdithiothreitol (DTT) broke the cross-links, liberated monomers andrestored the full TLR5 stimulatory activity of flagellin (FIG. 11 d,lane 8; and 11 e). As a control, flagellin monomers were treated withDSP, and this resulted in a chemically modified monomer whose migrationon SDS-PAGE was slightly retarded. This modified monomer retained fullbiological activity (FIG. 11 d, lanes 2 and 4; and 11f). These resultsdemonstrate that monomeric flagellin rather than filamentous flagellinstimulates TLR5, and that the flagellin TLR5 recognition site isinaccessible within the filament.

TLR5 also was demonstrated to physically interact with flagellin. Inthis regard, we investigated whether flagellin could precipitate TLR5.CHO cells transfected with V5-tagged TLR5 or TLR2 specifically recognizeflagellin or bacterial lipopeptide, respectively (FIG. 12 a). Flagellinwas purified from S. typhimurium, biotinylated and incubated with eitherTLR5 or TLR2 transfected CHO cells (FIGS. 12 b and c). Cells were eitherlysed and then incubated with biotinylated flagellin for 30 min on ice(4° C.) to examine flagellin-TLR interactions in cell lysates, orpre-incubated with biotinylated flagellin (37° C.) for 30 minutes andthen lysed to examine flagellin-TLR interactions in intact cells.Biotinylated flagellin and any associated proteins were affinitypurified from the lysates with streptavidin beads. Bound proteins wereeluted by boiling in SDS-PAGE sample loading buffer, and a immunoblotwas done to identify molecules bound to the beads.

Precipitation of TLR5 was performed with biotinylated bacterialflagellin. Briefly, purified flagellin was biotinylated with EZ-LinkSulfo-NHS-LC-Biotin (Pierce) and the buffer was changed back to PBS. CHOcells were transiently transfected with V5-tagged mTLR5, or mTLR2. After24 hours intact cells were incubated with biotinylated flagellin for 30min at 37° C., lysed, and nuclei cleared by 5 min centrifugation at3000×g, or lysed cells were cleared of nuclei, and the cleared lysatewas incubated with 10 ug/ml biotinylated flagellin for 30 min at 4° C.The lysates were next incubated with streptavidin agarose for 30 min at4° C., and the avidin-cleared supernatant was collected. The avidinbeads were washed extensively with PBS, and the purified proteins wereeluted by boiling in SDS-PAGE loading buffer. Cell equivalent portionsof the purification were separated by SDS-PAGE, and the V5-taggedproteins were identified by immunoblot, using the anti-PK antibody(Serotec), and a rabbit anti-mouse horse radish peroxidase secondary(Jackson Immunolabs).

FIG. 12(a) shows CHO K1 cells transfected with either V5-tagged mouseTLR5 or mouse TLR2, and an NF-kB luciferase reporter. Transfected cellswere stimulated with medium (control), LPS (100 ng/ml), bacteriallipopeptide (bLP, 300 ng/ml), or FliC (300 ng/ml), and the foldinduction of luciferase activity was quantified after 4 h. FIGS. 12(b)and (c) show V5-tagged mouse TLR5 and TLR2 transfected CHO K1 cellsincubated biotinylated flagellin either as intact cells for 30 min at37° C., or as cell lysates for 30 min at 4° C. The lysates (lanes 1 and4) were next incubated with streptavidin agarose beads for 30 min at 4°C., the avidin beads were collected by centrifugation, and the residualsupernatant was collected (avidin-cleared, lanes 2 and 5). The avidinbeads were washed extensively (avidin-purified, lanes 3 and 6). Cellequivalent portions were loaded in each lane, and mouse TLR5 and TLR2were detected by immunoblotting for the V5 epitope tag. CHO TLR5 lysateswere also incubated with biotinylated WT or the I411A FliC mutant,precipitated with streptavidin beads, and immunoblotted for the V5epitope tag (b lanes 7 and 8). Insertion mutants also were tested asdescribed previously using CHO K1 cells expressing human TLR5 (FIG. 12d) or mouse TLR5 (FIG. 12 e). Data shown is representative of at leastthree independent experiments. The amount of sonicated bacterial cellsused to stimulate approximately 10⁵ CHO cells in a 200 ul volume isindicated on the x-axis. The bacterial cells tested are shown in thelegend, and expressed either no FliC (BC379), (WT), the G166 insertionmutant (G166i::31), or the T168 insertion mutant (T168i::31).

The above results demonstrate that TLR5 specifically bound tobiotinylated flagellin (FIG. 12 b), whereas no association of flagellinwith TLR2 could be demonstrated (FIG. 12 c). In addition, the I411Amutation markedly reduced association of flagellin with TLR5 in celllysates (FIG. 12 b, lane 8). Because these experiments were done usingwhole cells, or cell lysates, the possibility remains that additionalfactors contributed and that the interaction of flagellin and TLR5 wasindirect. Although this is possible, we found that mouse and human TLR5discriminated between different flagellin insertion mutants whentransfected into the same cell line (FIGS. 12 d and e). Mouse TLR5recognized flagellin insertion mutants after residues 166 and 168,whereas human TLR5 did not. The species-specific differences in TLR5recognition of flagellin suggest that there is a direct interactionbetween flagellin and TLR5, since any additional factors were common inthis experimental system. Flagellar filaments are comprised of monomersof flagellin that stack together to form long protofilaments, 11 ofwhich wrap together to form the filament (Samatey, F. A. et al., Nature,410: 331-337 (2001)) and (Yonekura, K., et al., Nature, 424: 643-650(2003)).

Axial interactions occur between the concave surface (residues 56-69,and 132-151) of one monomer and a convex surface (89-107, 315, and408-409) of the underlying monomer to form the protofilament (Samatey,F. A. et al., Nature, 410: 331-337 (2001)). Overall 10 of the 13mutations that affect TLR5 recognition also affect motility, 10 of the19 mutations that affect motility also affect TLR5 recognition, and allmutations that we made in the convex surface reduce motility and/or TLR5recognition. Eleven residues in this convex surface were not testedbecause they are non-conserved, alanine or glycine, or have sidechainsthat are buried (Samatey, F. A. et al., Nature, 410: 331-337 (2001)).Residues involved in TLR5 recognition and motility overlap with theconvex surface involved in axial intermolecular contact between monomersin the protofilament. The combination of structural and functionalstudies demonstrates that residues R90, L94 and Q97 form a central coreof the flagellin structure that is critical for protofilament assembly,bacterial motility, as well as TLR5 recognition.

Protofilaments in the flagellar filament have the ability to convertbetween different helical states (left-handed (L) and right-handed (R)).The ability of the filaments to switch between L and R states isnecessary for propulsion and tumbling activity that is generated bycounterclockwise or clockwise rotation of the flagellar motor (Berg, H.C., et al., Nature 245: 380-382 (1973)) and (Larsen, S. H., et al.,Nature 249: 74-77 (1974)). When all of the filaments are locked intoeither the L or R state, the flagellum is straight and non-motile(Yamashita, I. et al., Nat. Struct. Biol., 5: 125-132 (1998)). TLR5recognition and motility are affected by additional mutations that areadjacent to, but outside the convex surface. Many of the mutations inthese residues have the most profound effect on bacterial motility. Someof the mutations that lie outside of the axial contact surface mayaffect this switching function by altering axial intermolecularinteractions, as has been shown for a flagellin D107E mutant (Kamiya,R., Asakura, et al., Nature, 286: 628-630 (1980)). Other mutations alterinteractions with their lateral partners (Samatey, F. A. et al., Nature,410: 331-337 (2001)) and (Yonekura, K., et al., Nature, 424: 643-650(2003)).

The alanine point mutations have a more profound effect on bacterialmotility than on TLR5 recognition. Of the 22 mutations, 8 reduce, and 11completely abrogate bacterial motility. None of the alanine mutationscompletely abrogates TLR5 recognition, and each of the 13 individualmutations that reduced TLR5 recognition did so by approximately 2-30fold. This effect on TLR5 recognition was relatively small compared tothe respective effect on bacterial motility, and we predict that mostindividual point mutations will not profoundly reduce innate immunerecognition of flagellin. The I411A mutation has the most profoundeffect on TLR5 recognition. The I411 sidechain is buried in the D1domain directly under the α-helix containing core residues R90, L94 andQ97, and likely exerts its effect by indirectly altering theconformation of several overlying residues. Thus, TLR5 recognizes acombinatorial surface on flagellin that is determined by the sum of alarge group of residues, and is somewhat permissive to variation inamino acid content within this site. The permissive nature of TLR5recognition of flagellin allows TLR5 to recognize a broad range ofbacterial flagellin molecules. In contrast, many point mutations destroybacterial motility, and thus the structural requirements for flagellarmotility are more rigid. These differences may explain why bacteria ingeneral have failed to evade TLR5 recognition. Such a feat would mostlikely require a very complex series of mutations in at least twodiscrete sites of the flagellin molecule (the convex/TLR5 site and theconcave site) that in sum would destroy TLR5 recognition, andsimultaneously compensate for any potential loss in motility.

The above results demonstrate that TLR5 recognizes flagellin monomersrather than filamentous flagellin, as the TLR5 recognition site is notaccessible in filaments, and thus flagellin filaments do not induce TLR5aggregation. TLR5 recognition of monomeric flagellin also has importantimplications for the recognition of bacterial flagellin during naturalinfections. Although it is likely that physical forces and chemicalfactors at the sites of bacterial infection would be capable ofliberating monomeric flagellin for TLR5 recognition, in many instancesthe predominant form of flagellin would be the flagellar filament, whichis anchored to the bacterium. This observation indicates that cellularrecognition of flagellin requires breaking down the filament to disperseat least a portion of the flagellin into its monomeric form. One meansto accomplish this result would be through phagocytosis, where theflagellar filaments would be exposed to an acidic environment thatpromotes filament depolymerization. In addition, epithelial cells arealso capable of recognizing flagellin, and models of polarized epitheliasuggest that apical infection of epithelial cells leads to thetranslocation of flagellin to the basolateral surface, where it isrecognized by TLR5 (Gewirtz, A. T., J. Immuno., 167: 1882-1885 (2001)).The apical uptake and basolateral translocation requires that flagellinis attached (as the filament) to a bacterium capable of invading thecell (Gewirtz, A. T. et al., J. Clin. Invest., 105: 79-92 (2000)). Ourstudies reveal an unrecognized and important step in the flagellinrecognition process. During uptake and translocation of flagellin, thefilament must undergo depolymerization to afford recognition by TLR5.

Recognition of flagellin by TLR5 also is sensitive; TLR5 can detectflagellin at concentrations of less than 100 fM. In addition, the aboveresults indicate that TLR5 most likely directly interacts with flagellinand that species-specific differences in TLR5 sequence dictate finespecificity for flagellin molecules, as is hypothesized for TLR4recognition of LPS (Lien, E. et al., J. Clin. Invest. 105: 497-504(2000); Poltorak, A., et al, Proc. Natl. Acad. Sci. USA, 97: 2163-2167(2000); and Hajjar, A. M., et al., Nat. Immunol. 3: 354-359 (2002)) andTLR9 recognition of CpG DNA (Bauer, S. et al., Proc. Natl. Acad. Sci.USA, 98: 9237-9242 (2001); and Takeshita, F. et al., J. Immunol., 167:3555-3558 (2001).

The recognition of flagellin by both plants and mammals suggests thatthis recognition is an evolutionarily ancient immune adaptation. Theprotein is recognized in plants by FLS2, a member of a family ofresistance genes. Other than sharing the common feature of anextracellular leucine rich repeat (LRR) domain, there is no significantamino acid similarity that suggests an evolutionary relationship betweenFLS2 and TLR5 (data not shown). Like TLR5, FLS2 utilizes the LRR domainfor detecting flagellin, although, TLR5 recognizes a conserved site onflagellin that is structurally distinct from the site recognized by FLS2(Felix, G., Duran, et al., Plant J., 18: 265-276 (1999)). Thus, plantsand animals have independently evolved LRR receptors that recognizebacterial flagellin, and activate host defense mechanisms.

A common prediction for PAMPs is that they are highly conserved andfunctionally essential, since they resist the evolutionary pressureimposed upon them by immune systems ranging from plants to animals. Theabove results directly address this prediction, and for the first timesubstantiate in molecular detail the exquisite ability of the innateimmune system to target a microbial structural unit that is functionallyrequired for bacterial fitness. The detailed understanding of theTLR5-flagellin interaction will provide insight into this very specificaspect of host-pathogen interactions, as well as permit rational designof novel immunomodulatory drugs and the engineering of flagellinproteins for vaccination.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention.

1. An immunomodulatory flagellin peptide comprising substantially thesame amino acid sequence GALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAE ITQ (SEQID NO:44), or a modification thereof, and having toll-like receptor 5(TLR5) binding.
 2. The flagellin peptide of claim 1, further comprisingsubstantially the same amino acid sequenceTQFSGVKVLAQDNTLTIQVGANDGETIDIDLKQINS QTLGLDTL (SEQ ID NO:45);EGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVNG (SEQ ID NO:46) orMAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQS (SEQ ID NO:47), or amodification thereof.
 3. The flagellin peptide of claim 1, furthercomprising TLR5 stimulating activity.
 4. The flagellin peptide of claim1, further comprising an ADCC targeting molecule.
 5. An immunomodulatoryflagellin peptide comprising substantially the same amino acid sequenceLQKIDAALAQVDTLRSDLGAVQNRFNSAITNL (SEQ ID NO:48), or a modificationthereof, and having toll-like receptor 5 (TLR5) binding.
 6. Theflagellin peptide of claim 5, further comprising substantially the sameamino acid sequence TLRSDLGAVQNRFNSAITNLGNTVNNLSS (SEQ ID NO:49) orEQAAKTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLSS (SEQ ID NO:50), or amodification thereof.
 7. The flagellin peptide of claim 5, furthercomprising TLR5 stimulating activity.
 8. The flagellin peptide of claim5, further comprising an ADCC targeting molecule.
 9. An immunomodulatoryflagellin peptide comprising substantially the same amino acid sequenceGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAE ITQ (SEQ ID NO:44) andsubstantially the same amino acid sequenceLQKIDAALAQVDTLRSDLGAVQNRFNSAITNL (SEQ ID NO:48), or a modificationthereof, and having toll-like receptor 5 (TLR5) binding.
 10. Theflagellin peptide of claim 9, further comprising substantially the sameamino acid sequence TQFSGVKVLAQDNTLTIQVGANDGETIDIDLKQINS QTLGLDTL (SEQID NO:45); EGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVNG (SEQ IDNO:46), MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQS (SEQ ID NO:47),TLRSDLGAVQNRFNSAITNLG NTVNNLSS (SEQ ID NO:49) orEQAAKTTENPLQKIDAALAQVDTLRSDLGAVQ NRFNSAITNLGNTVNNLSS (SEQ ID NO:50), ora modification thereof.
 11. The flagellin peptide of claim 9, furthercomprising TLR5 stimulating activity.
 12. The flagellin peptide of claim9, further comprising an ADCC targeting molecule.
 13. The flagellinpeptide of claim 9, further comprising a linker joining flagellin aminoterminal residues 79-117 and flagellin carboxyl terminal residues408-439.
 14. A method of inducing an antigen-specific immune response inan individual comprising, administering to an individual an immunogenicamount of a vaccine, said vaccine having an antigen and animmunomodulatory flagellin peptide comprising a flagellin peptide ofclaims 1, 5 or
 9. 15. The method of claim 14, wherein said antigen isselected from the group consisting of polypeptides, polysaccharides,pathologically aberrant cells and bacteria.
 16. A method of inducing aTLR5-mediated response, comprising administering to a TLR5-containingcell an effective amount of an immunomodulatory flagellin peptide ofclaims 1, 5 or
 9. 17. The method of claim 16, wherein said TLR5-mediatedresponse is TLR5-induced modulation of cytokine amount or activity. 18.The method of claim 16, wherein said TLR5-mediated response isTLR5-induced increase in an amount of a cytokine selected from the groupconsisting of TNFa, IL-1 and IL-6.
 19. The method of claim 16, whereinsaid TLR5-mediated response is TLR5-induced NF-kB activity.
 20. A methodof inducing an immure response in an individual having a pathologicalcondition, comprising administering to said individual an immunogenicamount of an immunomodulatory flagellin peptide of claims 1, 5 or
 9. 21.The method of claim 20, wherein said pathological condition is selectedfrom the group consisting of proliferative disease, autoimmune disease,infectious disease and inflammatory disease.
 22. The method of claim 20,wherein said immunomodulatory flagellin peptide further comprises anADCC targeting molecule.
 23. A method of modulating an immune responsein an individual having a pathological condition, comprisingadministering to said individual a combination of an immunogenic amountof an immunomodulatory flagellin peptide of claims 1, 5 or 9, and animmunomodulatory molecule.
 24. The method of claim 23, wherein saidimmunomodulatory molecule is an antibody, cytokine or growth factor. 25.The method of claim 23, wherein said immunomodulatory flagellin peptidefurther comprises an ADCC targeting molecule.
 26. The method of claim23, wherein said pathological condition is selected from the groupconsisting of proliferative disease, autoimmune disease, infectiousdisease and inflammatory disease.
 27. A screening composition,comprising: (a) a flagellin peptide of claims 1, 5 or 9, and (b) a TLR5polypeptide or modification thereof, having a TLR5 activity.
 28. Thecomposition of claim 27, further comprising a detectably labeledflagellin peptide.
 29. The composition of claim 27, wherein said TLR5polypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:6 and SEQ ID NO:8, or a modification or fragmentthereof having a TLR5 activity.
 30. A method of screening for a TLR5ligand, agonist or antagonist, comprising: (a) contacting a TLR5 with acandidate compound in the presence of an immunomodulatory flagellinpeptide of claims 1, 5 or 9 under conditions wherein binding of saidimmunomodulatory flagellin peptide to said TLR5 produces a predeterminedsignal; (b) determining the production of said predetermined signal inthe presence of said candidate compound, and (c) comparing saidpredetermined signal in the presence of said candidate compound with apredetermined signal in the absence of said candidate compound, whereina difference between said predetermined signals in the presence andabsence of said candidate compound indicates that said compound is aTLR5 ligand, agonist or antagonist.
 31. The method of claim 30, whereinsaid predetermined signal is selected from the group consisting ofpolypeptide amount, polypeptide activity and transcriptional activity.32. The method of claim 30, wherein said predetermined signal is amountof a cytokine selected from the group consisting of TNFα, IL-1 and IL-6.33. The method of claim 31, wherein said transcriptional activity isNF-kB activity.